钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位北大核心CSCD

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻(Spirulina platensis)FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻（Spirulina platensis）FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

叶绿体分裂相关基因NtFtsZ2-1在大肠杆菌中的表达与定位

FtsZ蛋白在细菌的分裂中担任着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。我们为了研究烟草FtsZ蛋白与大肠杆菌FtsZ蛋白的异同,构建了烟草全长ftsZ2-1与绿色荧光蛋白EGFP的融合表达质粒并转化大肠杆菌JM109。融合表达质粒的过量表达导致宿主菌形成了丝状菌体。通过荧光显微镜观察发现NtFtsZ2-1-EGFP融合蛋白沿着宿主菌体的纵轴方向有规律地聚集成荧光点或荧光带,说明烟草FtsZ2-1蛋白能够识别宿主菌内分裂位点的定位信号并参与其细胞分裂复合物的组装。     

衣藻CrFtsZ2-GFP融合蛋白在E.coli中的表达及其定位

FtsZ蛋白在细菌的分裂中担任着重要作用 ,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。胞内FtsZ蛋白浓度的异常升高或降低均可阻断正常的细胞分裂过程进而形成分裂异常的丝状菌体。为了研究衣藻FtsZ蛋白的生物学活性 ,构建了衣藻CrFtsZ2cDNA全长与绿色荧光蛋白基因egfp的融合表达质粒 ,并对其在大肠杆菌中的表达与定位做了初步分析。在大肠杆菌JM10 9中 ,融合表达质粒的过量表达导致宿主菌形成了丝状菌体 ,通过荧光显微镜观察发现CrFtsZ2 EGFP融合蛋白沿着宿主菌体的纵轴方向有规律地聚集成荧光点或荧光带 ,暗示衣藻CrFtsZ2蛋白能够识别宿主菌内分裂位点的定位信号并参与其细胞分裂过程 ,初步验证了衣藻CrFtsZ2蛋白的功能。     

FtsZ蛋白同源性分析在乳酸菌系统学研究中的应用

FtsZ是一种广泛存在于细菌和古菌中的结构保守的蛋白质,在细胞分裂的过程中起关键的作用。通过PCR扩增FtsZ基因的一段800bp的核苷酸,构建了干酪乳杆菌-片球菌及相关乳酸菌的FtsZ蛋白系统发育树。将此系统树和16SrDNA系统树比较发现二者的拓扑结构非常相似。在两个基因系统树中,片球菌与乳杆菌的种均显示了较近的亲缘关系,而与其它球状的乳酸菌,如链球菌和肠球菌的亲缘关系较远。研究还表明FtsZ蛋白序列的分辨率高于16SrDNA,更适用于乳酸菌种间的系统分类研究。     

衣藻CrMinD基因的网上克隆及其进化分析

细菌细胞正常分裂时,在其中部形成介导细胞分裂的环状复合物结构。该环状复合物至少由10多种蛋白组成。其中,FtsZ蛋白最早在细胞中部组装成环状结构Z环,其他分裂相关蛋白再先后与Z环相结合,行使其分裂功能。Fts蛋白为原核细胞骨架蛋白,与真核生物的微管蛋白具有共同的进化祖先。在大肠杆菌细胞中共有三个潜在的细胞分裂位点,一在中部,另外两个分部在两极。正常情况下仅有中部的分裂位点得到应用。FtsZ环正确定位于细胞中部的潜在分裂位点与MinD蛋白密切相关。当minD基因突变时FtsZ蛋白则在细胞两极组装成Z环,最终导致细胞分裂异常,产生不含基因组的小细胞(Mincell)。       

衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E．coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。     

靶向FtsZ的细胞分裂抑制剂的研究进展

细菌耐药性的日益凸显严重威胁着人类健康。传统的筛选方法已经难以筛选到新的抗生素。运用新的技术去开发新的抗生素迫在眉睫。FtsZ(filamentous temperature-sensitive protien Z)作为一种广泛存在于细菌中的重要的细胞分裂蛋白目前广受关注。该文简要概述了FtsZ在细胞分裂中的作用,靶向FtsZ的细胞分裂抑制剂筛选模型的建立,以及已经筛选获得的一些具有生理活性的FtsZ抑制剂。     

VicRK(YycFG)双组分调节系统

细菌双组分调节系统,或称之为双组分信号转导系统,是细菌感应外界多变环境,维持自身存活和生长繁衍的重要感应系统.在这些调节系统中,最早发现于枯草芽孢杆菌的VicRK(YycFG)系统因与细胞存活密切相关而倍受关注.该系统存在于少数低G+C含量的革兰氏阳性菌中,包括金黄色葡萄球菌和肺炎链球菌等致病菌,高度保守.许多证据显示,VicRK(YycFG)具有调控细胞壁合成与代谢、胞膜完整、细胞分裂、脂类代谢、多糖合成与被膜形成以及细菌毒力等多种功能,参与细胞的生长、分裂与感染.该系统异常可导致细菌生活力严重下降,甚至死亡,因而成为防治该类病原菌的重要靶标.     

细菌分裂Z环定位的调控方式

细菌细胞的分裂调控机制一直是人们研究的热点.在细胞中部形成一个隔膜,这一看似简单的过程是一个多因子参与调控的过程.Z环(FtsZ ring)是分裂体的支架,Z环形成的位置不仅是隔膜形成的位置还决定着细胞分裂位点,Z环在不正确的位置形成会导致细胞不均等分裂.目前研究已经发现了细胞分裂的多种调控包括Min系统、类核闭塞、MipZ蛋白,通过不同机制可以有效避免Z环的组装,从而阻止了分裂体在不正确的位置形成.就目前研究的Z环形成的过程以及影响Z环定位的调控机制作一综述.     

Cell Shape Can Mediate the Spatial Organization of the Bacterial Cytoskeleton

The bacterial cytoskeleton guides the synthesis of cell wall and thus regulates cell shape. Because spatial patterning of the bacterial cytoskeleton is critical to the proper control of cell shape, it is important to ask how the cytoskeleton spatially self-organizes in the first place. In this work, we develop a quantitative model to account for the various spatial patterns adopted by bacterial cytoskeletal proteins, especially the orientation and length of cytoskeletal filaments such as FtsZ and MreB in rod-shaped cells. We show that the combined mechanical energy of membrane bending, membrane pinning, and filament bending of a membrane-attached cytoskeletal filament can be sufficient to prescribe orientation, e.g., circumferential for FtsZ or helical for MreB, with the accuracy of orientation increasing with the length of the cytoskeletal filament. Moreover, the mechanical energy can compete with the chemical energy of cytoskeletal polymerization to regulate filament length. Notably, we predict a conformational transition with increasing polymer length from smoothly curved to end-bent polymers. Finally, the mechanical energy also results in a mutual attraction among polymers on the same membrane, which could facilitate tight polymer spacing or bundling. The predictions of the model can be verified through genetic, microscopic, and microfluidic approaches.     

Assembly of the MreB‐associated cytoskeletal ring of Escherichia coli

The Escherichia coli actin homologue MreB is part of a helical cytoskeletal structure that winds around the cell between the two poles. It has been shown that MreB redistributes during the cell cycle to form circumferential ring structures that flank the cytokinetic FtsZ ring and appear to be associated with division and segregation of the helical cytoskeleton. We show here that the MreB cytoskeletal ring also contains the MreC, MreD, Pbp2 and RodA proteins. Assembly of MreB, MreC, MreD and Pbp2 into the ring structure required the FtsZ ring but no other known components of the cell division machinery, whereas assembly of RodA into the cytoskeletal ring required one or more additional septasomal components. Strikingly, MreB, MreC, MreD and RodA were each able to independently assemble into the cytoskeletal ring and coiled cytoskeletal structures in the absence of any of the other ring components. This excludes the possibility that one or more of these proteins acts as a scaffold for incorporation of the other proteins into these structures. In contrast, incorporation of Pbp2 required the presence of MreC, which may provide a docking site for Pbp2 entry.     

FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli

Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis, and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance. Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern. The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings. Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins. The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD. The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.     

Increasing complexity of the bacterial cytoskeleton

Bacteria contain cytoskeletal elements involved in major cellular processes including DNA segregation and cell morphogenesis and division. Distant bacterial homologues of tubulin (FtsZ) and actin (MreB and ParM) not only resemble their eukaryotic counterparts structurally but also show similar functional characteristics, assembling into filamentous structures in a nucleotide-dependent fashion. Recent advances in fluorescence microscopic imaging have revealed that FtsZ and MreB form highly dynamic helical structures that encircle the cells along the inside of the cell membrane. With the discovery of crescentin, a cell-shape-determining protein that resembles eukaryotic intermediate filament proteins, the third major cytoskeletal element has now been identified in bacteria as well.     

A growing family: the expanding universe of the bacterial cytoskeleton

Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes.     

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.     

YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli

All free-living bacteria carry the toxin-antitoxin (TA) systems controlling cell growth and death under stress conditions. YeeU-YeeV (CbtA) is one of the Escherichia coli TA systems, and the toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. Here, we demonstrate that the antitoxin, YeeU, is a novel type of antitoxin (type IV TA system), which does not form a complex with CbtA but functions as an antagonist for CbtA toxicity. Specifically, YeeU was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Surprisingly, YeeU neutralized not only the toxicity of CbtA but also the toxicity caused by other inhibitors of MreB and FtsZ, such as A22, SulA and MinC, indicating that YeeU-induced bundling of MreB and FtsZ has an intrinsic global stabilizing effect on their homeostasis. Here we propose to rename YeeU as CbeA for cytoskeleton bundling-enhancing factor A.     

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.     

Sublethal High Hydrostatic Pressure Treatment Reveals the Importance of Genes Coding Cytoskeletal Protein in Escherichia Coli Morphogenesis

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.