EXPRESSION AND LOCATION OF CRFTSZ2 GENE FROM CHLAMYDOMONAS REINHARDTII IN E. coli

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.