衣藻CrFtsZ2-GFP融合蛋白在E.coli中的表达及其定位
EXPRESSION AND LOCATION OF CRFTSZ2 GENE FROM CHLAMYDOMONAS REINHARDTII IN E. coli
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摘要: 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CrFtsZ2 cDNA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JM109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.Abstract: In the current model for bacterial cell division,the essential cell division protein FtsZ forms a ring that marks the division plane,creating a cytoskeletal framework for the subsequent action of other proteins.FtsZ protein localizes to the cell midpoint very early in cytokinesis. FtsZ is probably present in all eubacteria,archaea. Immunoelectron microscopy of thin sections of bacterial cells demonstrated that FtsZ polymerizes to form a circumferential ring at the mid-cell division site,constricting at the leading edge of the invaginating septum that eventually separates the two daughter cells. The deficiency or overexpression of FtsZ inhibits the normal cell division, leading filamentation cells in E. coli. Consistent with its cytoskeletal role,FtsZ has certain properties in common with the eukaryotic cytoskeletal protein tubulin. Like tubulin,purified FtsZ binds and hydrolyzes GTP and polymerizes to form long tubules in a GTP-dependent manner. Because plastids have arisen from an endosymbiotic event between a primitive eukaryotic cell and a photosynthetic prokaryote,bacterial cell division has been used as a paradigm to dissect plastid division. It is now clear that plant chloroplasts maintain the division apparatus from their bacterial progenitors. Here,we use green fluorescent protein(GFP)to tag CrFtsZ2,which was encoded by CrFtsZ2 gene from a unicellular green alga Chlamydomonas reinhardtii,so that their structure and location can be visualized in living E. coli cells. To visualize FtsZ proteins in living E.coli cells,a reconstructed plasmid,named pLGZ2,was constructed that contained GFP fusions to full-length CrftsZ cDNA. Expression of the fusions was under control of the lac promoter operator on the plasmids and lacⅠq on the same plasmid. The localization and structure of FtsZ-GFP were characterized in living cells taken from freshly grown liquid cultures with IPTG for 5 hours. The phenotypes of the cells contain pLGZ2,showing normal-length,were similar with the wild type when without IPTG. After inducing with IPTG,however,E.coli cells with higher levels of CrFtsZ-GFP were inhibited for cell division and often exhibited bright fluorescent dots that spanned the length of the filamentous cells. The spacing of the dots was remarkably uniform in any given cell,but ranged anywhere between 2 um and as much as 5μm,depending on the cell. The dots were located consistently suggesting that this form of CrFtsZ-GFP was probably localizing to potential division site(PDS). These results implied that CrFtsZ2 protein from Chlamydomonas reinhardtii could still recognize the signals for division site positioning in bacteria and take part in the bacterial division complexity. Furthermore,these result not only provide the direct evidence to support the endosymbiosis hypothesis,but also establish a feasible foundation for further studies of in vitro polymerization an in vivo subcellular localization of eukaryotic FtsZ.
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Keywords:
- FtsZ protein /
- Chlamydomonas reinhardtii /
- Confusion expression /
- Cell division
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[1] Hirota Y.Thermosensitive mutants of E.coli affected in the process of DNA synthesis and cell division[J].Cold Spring Harbor Symp Quant Biol, 1968,33: 677[2] Lutkenhaus J F,Wolf-Watz H,Donachie W D. Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus(ftsZ)[J]. J Bacteriol,1980,142(2):615-620[3] Lutkanhaus J F,Addinall S G. Bacterial cell division and the Z ring[J]. Ann rev Biochem,1997,66:93-116[4] Bi E F,Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli[J]. Nature,1991,354(6349):161-164[5] Addinall S G,Bi E,Lutkenhaus J. FtsZ ring formation in fts mutants[J].J Bacteriol,1996,178(13):3877-3884[6] Osteryoung K W,Stokes K D,Rutherford S M,et al. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ[J]. Plant Cell.1998,10:1991-2004[7] Wang D,Kong D D. Ju C L .et al. Effects of tobacco plastid division genes NtFtsZ1-1 and NtFtsZ1-2 on the division and morphology of chloroplasts[J]. Acta Botan Sin,2002,44(7)∶838-844[8] He Y K,Zhu C F,Wang D,et al. Clonging of plastid division gene GIFtsZ from Gentiana lutea and its expression during petal development[J]. Prog Nat Sci,2002,12(8):592-597(何奕昆,朱长甫,王东等. 龙胆GIFtsZ基因的克隆及其在花瓣发育过程中的表达. 自然科学进展,2002,12(8):821-825)[9] Reski R.Rings and networks:the amazing complexity of FtsZ in chloroplasts[J]. Trends Plant Sci,2002,7(3):103-105[10] Strepp R,Scholz S,Kruse S,et al. Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ,an ancestral tubulin[J]. Proc Natl Acad Sci USA,1998,95:4368-4373[11] McAndrew R S,Froehlich J E,Vitha S,et al. Colocalization of plastid division proteins in the chloroplast stromal compartment establishes a new functional relationship between FtsZ1 and FtsZ2 in higher plants[J]. Plant Physiol,2001,127:1656-1666[12] Wakasugi T,Nagai T,Kapoor M,et al. Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris:the existence of genes possibly involved in chloroplast division[J]. Proc Natl Acad Sci USA,1997,94:5967-5972[13] Martin W,Stoebe B,Goremykin V,et al. Gene transfer to the nucleus and the evolution of chloroplasts[J]. Nature,1998,393:162-165[14] Douglas,S E,Penny S L. The plastid genome of the cryptophyte alga,Guillardia theta:complete sequence and conserved synteny groups confirm its common ancestry with red algae[J].J Mol Evol,1999,48(2):236-244[15] Glockner G,Rosenthal A,Valentin K. The structure and gene repertoire of an ancient red algal plastid genome[J]. J Mol Evol,2000,51:382-390[16] Martin W,Herrmann R G. Gene transfer from organelles to the nucleus:how much,what happens, and Why [J]? Plant Physiol,1998,118:9-17[17] Hu Y,Wang D,Kong D D,et al. Cloning and phylogenetic analysis of CrFtsZ2 in Chlamydomonas reinhardtii[J]. Chin J Biochem Mol Biol,2003,19(1):127-132(胡勇,王东,孔冬冬,等. 衣藻质体分裂相关基因CrFtsZ2的克隆及其进化分析. 中国生物化学与分子生物学报,2003,19(1):127-132)[18] Baumann L,Baumann P. Characterization of ftsZ,the cell division gene of Buchnera aphidcola (endosymbiont of aphids)and detection of the product [J]. Curr Microbiol,1998,36(2):85-89[19] Salimnia H,Radia A,Bernatchez S,et al. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae[J]. Arch Microbiol,2000,173(1):10-20[20] Ma X,Ehrhardt D W,Margolin W. Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein[J]. Proc Natl Acad Sci USA,1996,93(23):12998-13003[21] Osteryoung K W,Vierling E. Conserved cell and organelle division[J]. Nature,1995,376,(6540):473-474[22] Kuroiwa T,Kuroiwa H,Sakai A,et al. The division apparatus of plastids and mitochondria[J]. Int Rev Cytol,1998,181:1-41[23] Kuroiwa T. The discovery of the division apparatus of plastids and mitochondria[J]. J Electron Microsc,2000,49(1):123-134 Hirota Y.Thermosensitive mutants of E.coli affected in the process of DNA synthesis and cell division[J].Cold Spring Harbor Symp Quant Biol, 1968,33: 677[2] Lutkenhaus J F,Wolf-Watz H,Donachie W D. Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus(ftsZ)[J]. J Bacteriol,1980,142(2):615-620[3] Lutkanhaus J F,Addinall S G. Bacterial cell division and the Z ring[J]. Ann rev Biochem,1997,66:93-116[4] Bi E F,Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli[J]. Nature,1991,354(6349):161-164[5] Addinall S G,Bi E,Lutkenhaus J. FtsZ ring formation in fts mutants[J].J Bacteriol,1996,178(13):3877-3884[6] Osteryoung K W,Stokes K D,Rutherford S M,et al. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ[J]. Plant Cell.1998,10:1991-2004[7] Wang D,Kong D D. Ju C L .et al. Effects of tobacco plastid division genes NtFtsZ1-1 and NtFtsZ1-2 on the division and morphology of chloroplasts[J]. Acta Botan Sin,2002,44(7)∶838-844[8] He Y K,Zhu C F,Wang D,et al. Clonging of plastid division gene GIFtsZ from Gentiana lutea and its expression during petal development[J]. Prog Nat Sci,2002,12(8):592-597(何奕昆,朱长甫,王东等. 龙胆GIFtsZ基因的克隆及其在花瓣发育过程中的表达. 自然科学进展,2002,12(8):821-825)[9] Reski R.Rings and networks:the amazing complexity of FtsZ in chloroplasts[J]. Trends Plant Sci,2002,7(3):103-105[10] Strepp R,Scholz S,Kruse S,et al. Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ,an ancestral tubulin[J]. Proc Natl Acad Sci USA,1998,95:4368-4373[11] McAndrew R S,Froehlich J E,Vitha S,et al. Colocalization of plastid division proteins in the chloroplast stromal compartment establishes a new functional relationship between FtsZ1 and FtsZ2 in higher plants[J]. Plant Physiol,2001,127:1656-1666[12] Wakasugi T,Nagai T,Kapoor M,et al. Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris:the existence of genes possibly involved in chloroplast division[J]. Proc Natl Acad Sci USA,1997,94:5967-5972[13] Martin W,Stoebe B,Goremykin V,et al. Gene transfer to the nucleus and the evolution of chloroplasts[J]. Nature,1998,393:162-165[14] Douglas,S E,Penny S L. The plastid genome of the cryptophyte alga,Guillardia theta:complete sequence and conserved synteny groups confirm its common ancestry with red algae[J].J Mol Evol,1999,48(2):236-244[15] Glockner G,Rosenthal A,Valentin K. The structure and gene repertoire of an ancient red algal plastid genome[J]. J Mol Evol,2000,51:382-390[16] Martin W,Herrmann R G. Gene transfer from organelles to the nucleus:how much,what happens, and Why [J]? Plant Physiol,1998,118:9-17[17] Hu Y,Wang D,Kong D D,et al. Cloning and phylogenetic analysis of CrFtsZ2 in Chlamydomonas reinhardtii[J]. Chin J Biochem Mol Biol,2003,19(1):127-132(胡勇,王东,孔冬冬,等. 衣藻质体分裂相关基因CrFtsZ2的克隆及其进化分析. 中国生物化学与分子生物学报,2003,19(1):127-132)[18] Baumann L,Baumann P. Characterization of ftsZ,the cell division gene of Buchnera aphidcola (endosymbiont of aphids)and detection of the product [J]. Curr Microbiol,1998,36(2):85-89[19] Salimnia H,Radia A,Bernatchez S,et al. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae[J]. Arch Microbiol,2000,173(1):10-20[20] Ma X,Ehrhardt D W,Margolin W. Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein[J]. Proc Natl Acad Sci USA,1996,93(23):12998-13003[21] Osteryoung K W,Vierling E. Conserved cell and organelle division[J]. Nature,1995,376,(6540):473-474[22] Kuroiwa T,Kuroiwa H,Sakai A,et al. The division apparatus of plastids and mitochondria[J]. Int Rev Cytol,1998,181:1-41[23] Kuroiwa T. The discovery of the division apparatus of plastids and mitochondria[J]. J Electron Microsc,2000,49(1):123-134
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