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Localization of Two GFP-tagged Tobacco Plastid Division
Protein NtFtsZs in Escherichia coli
WANG Dong1, KONG Dong-Dong1, JU Chuan-Li2, HU Yong2, HE Yi- Kun23, SUN Jing-San13
(1. Institute of Botany , The Chinese Academy of Sciences , Beijing 100093 ,China ; 2 . Department of Biology , Capital Normal University , Beijing 100037 , China)
Abstract : Two plastid division genes , NtFtsZ1 and Nt FtsZ2 isolated from Nicotiana tabacum L. were fused
with gfp and expressed in Escherichia coli. The regular localizations of full length NtFtsZs∶GFP along the fila2
mentous bacteria indicated that the NtFtsZs could recognize the potential division sites in E. coli and be poly2
merized with heterogeneous FtsZ from bacteria. The overexpression of NtFtsZs∶gfp inhibited the division of
host strain cells and resulted in the longfilamentous bacterial morphology. These results suggested that eukary2
otic ftsZs have similar function to their prokaryotic homologs. Meanwhile , the different deletions of motifs of
NtFtsZs are also employed to investigate the functions of these proteins in E. coli. The results showed that the
C-terminal domains of NtFtsZs were related to the correct localization of NtFtsZs in E. coli and the N-terminal
domains of NtFtsZs were responsible for the polymerization of homogeneous and heterogeneous FtsZ proteins.
The significance of these results in understanding the functions of NtFtsZs in plastid division were discussed.
Key words : Nicotiana tabacum ; plastid division gene ; Nt FtsZ ; GFP ; localization in Escherichia coli
FtsZis a conservative and primitive cytoskeleton pro2
tein found from the Escherichia coli temperature sensitive
mutant Z (filamentation temperature- sensitive Z , ftsZ).
During the cell division cycle , FtsZ assembles into a ring
structure at the division site before any other known cell
division proteins , and the constriction of the ring structure
results in cell division[1 ] . FtsZ is a GTP-binding protein
with GTPase activity and also shares some considerable
biochemical and structural similarities with eukaryotic cy2
toskeleton component tubulin , leading to the hypothesis
that eukaryotic cytoskeloton might be evolved from
prokaryotic cytoskeletal elements[ 1 - 3 ] . Compared with
other known cell division proteins , FtsZ emerges in the
early stage of cell division and plays an organizer to re2
cruit the other cell division proteins forming a putative
molecular apparatus dedicated to dividing the cell. Thus ,
FtsZ is the most important cell division protein in prokary2
otic cell division cycle hereto known[ 1 ,2 ] .
More recently , FtsZ also has been found in higher
plant[ 4 ] and its role in the plastid division has been estab2
lished preliminarily[ 5 ,6 ] . However , there are some differ2
ent views about the functional patterns of plant FtsZ in
control of pla stids division and/ or plastid shape maintain2
ing. In a moss Physcomitrella patens , FtsZ not only influ2
enced the plastid division[6 ] , but also possibly consisted
of a new subcellular structure-plastoskeleton[7 ] . In Ara2
bidopsis , FtsZs from different families seemly form a ring-
like structure located at two sides of membrane of plas2
tid[ 5 ,8] ; the ring structure is thought to be a reminiscence
of prokaryotic FtsZ ring. However , recent studies on a
unicellular eukaryotic red alga Cyanidioschyzon merolae
suggested that the FtsZ may not be the component of the
plastid- dividing rings , at least not be the component of
outer ring[9 ] . All the studies make the functional patterns
of plant FtsZs more confused.
Because of the importance and conservative function
of FtsZs in prokaryotic cell division , it provides an effi2
cient method to study heterogeneous FtsZ function in E.
coli [ 10] . To further understand the difference between
plant and prokaryotic FtsZs , here we report that two plas2
tid division proteins NtFtsZ1 and NtFtsZ2 , which are en2
coded by tobacco genomic genes and were tagged with
green fluorescent protein (GFP), localized in living bac2
terial cells in a visible method. The polymerization , lo2
calization and effects of fusion proteins on bacterial mor2
phology were worked on. Meanwhile , the effects of differ2
ent deletions of NtFtsZ proteins on the polymerization and
localization of fusion proteins in E. coli were also anal2
ysed.
1 Materials and Methods
1. 1 Materials
The plasmids containing full-length cDNA of
NtFtsZ1 and Nt FtsZ2 , cloning vector pBluescript ⅡKS
(+)and Escherichia coli strain JM109 are kept in our
laboratory. The GFPmut2 plasmid , which contained
gfpS65A ,V68L ,S72A mutants , was a gift from Prof. LI Jiu-Di
(Institute of Botany , the Chinese Academy of Sciences).
PCR primers were from Shanghai Sangon. DNAs were se2
quenced by TaKaRa (Dalian , China). Anti- GFP poly2
clonal antiserum was purchased from CLONTECH.
1. 2 Construction of expression vectors
The constructions of different expression vectors refer
to Fig. 1. All the constructs were made by PCR. The rel2
ative positions of primers in different constructs are shown
in Fig. 1. To ensure the fidelity of PCR amplification ,
Received: 2001207209 Accepted : 2001210219
Supported by the National Natural Science Foundation of China (39970356)and the Natural Science Foundation of Beijing (5992003).
3Author for correspondence. E-mail : < yhe @duke. edu ; sunjs @ns. ibcas. ac. cn > .
植 物 学 报
Acta Botanica Sinica 2002 ,44 (8):931 - 935
© 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.
Taq was replaced by pfu in the PCR reaction. The fusion
junctions were confirmed by DNA sequencing. To facili2
tate cloning and expression , all the fusion genes were in2
serted between the BamHⅠand Sac Ⅰsite of pBlue2
script ⅡKS (+)and under the control of lac promoter.
1. 3 General molecular manipulations were carried out
according to Molecular Cloning[ 11 ] .
1. 4 Western blotting
The preparation , separation and electroblotting of to2
tal proteins were conducted by the standard proce2
dures[11 ] . The correct expressions of fusion proteins were
confirmed by immunoblot analysis with anti- GFP polyclon2
al antiserum (Fig. 2).
1. 5 Microscopic techniques
In general , the transformed cells were cultured un2
der the conditions as described[12] and modified slightly.
Overnight growth of colonies on LB plate with ampicillin
were resuspended in 5μ
L LB and a equal volume of warm
0. 5 % low melting temperature agarose ,the mixture solu2
tion was immediately dropped on a glass slide and covered
with a cover glass. Microscopic observation and photogra2
phy were performed under Leica DMRE microscope. Im2
ages of bacterial morphology were obtained using differen2
tial interference contrast (Nomarski)optics. For GFP flu2
orescence microscopy , an FITC (excitation 455 - 495
nm , emission 512 - 575 nm)filter set was used and im2
ages were captured with a Leica DC200 Digital Camera.
All the images were assembled for publication using
Adobe Photoshop 5. 0.
2 Results
2. 1 Strategy of expression vector construction
All the known FtsZproteins contain two different do2
mains , the conservative N-terminal GTP binding domain
and the divergent C-terminal variation region (Fig. 1).
There are two special motifs of FtsZ protein existed in the
N-terminal conservative region , i. e. FTSZ-1 :
VIGVGGGGSNAVNRM (PROSITE PS01134)and FTSZ-
2 : FATAGMGGGTGS/ TGAAPV/ IV/ IA (PROSITE
PS01135).The FTSZ-2 also includes a typical tubulin
signature motif GGGTGS/ TG (PROSITE PS00277)and
its function had been studied in prokaryotic FtsZproteins ,
indicating that it is involved in the polymerization and the
GTPase activity of FtsZ. The deletion of FTSZ-2 disrupted
the normal polymerization of FtsZ and the formation of Z-
ring, and then the normal cell division cycle[ 1 ,2 ] . Site-
specific mutation examinations also confirmed that FTSZ-2
is important for GTPase activity of FtsZ protein[1 ,2 ,13] . On
the other hand , the function of FTSZ-1 still kept un2
known. The C-terminal domain of FtsZ protein had no ob2
vious sequence similarity among different FtsZ proteins ex2
cept for the extreme C-terminus[14]. Primary studies in
prokaryotic cells shows that the function of the C-terminal
region may be responsible for the interaction of FtsZ with
other cell division protein[ 14 ] . Based on the previous
studies , we constructed a series of expression vectors
(Fig. 1)and studied the functions of NtFtsZs in E. coli.
Fig. 1 . Schematic representation of the construction strategy of Nt Fts Zs : gfp fusion expression vectors.
Pairs of PCR primers were specific for NtFtsZ1 full-length cDNA (P1 and P2),NtFtsZ2 full-length cDNA (P3 and P4), C-terminal deletion
of NtFtsZs (P1 and P5 for NtFtsZ1 , P3 and P5 for NtFtsZ2), N-terminal deletion of NtFtsZs (P6 and P7 for Nt FtsZs
Δ
N1 , P8 and P9 for
NtFtsZs
Δ
N2), respectively. Purified plasmids containing NtFtsZ1 or NtFtsZ2 cDNA were used as the templates for PCR amplification.
932 植物学报 Acta Botanica Sinica Vol. 44 No. 8 2002
© 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.
Fig. 2 . Western blotting analyses of fusion proteins expression.
Analyzed were NtFtsZs∶GFP (lanes 1 and 5 ), NtFtsZs
ΔC∶GFP
(lanes 2 and 6), NtFtsZs
Δ
N1∶GFP (lanes 3 and 7), NtFtsZs
Δ
N2∶
GFP (lanes 4 and 8)and GFP only (lane C), respectively. The
calculated molecular weight of fusion protein was 73 , 70 , 69 kD for
full-length Nt FtsZs , C-terminal deletions and N-terminal deletions ,
respectively.
2. 2 Localization of the full length Nt Fts Zs ∶gfp in
E. coli
The full length N . tabacum NtFtsZ1 and NtFtsZ2
cDNAs lacking stop codons were fused in a frame with
gfpS65A ,V68L ,S72A gene and expressed in E. coli strain
JM109. The E. coli cells cultured on plates without IPTG
had no obvious changes (Fig. 3A), and displayed a simi2
lar morphology with that of the control cells (Fig. 3B).
When the cells were cultured on plates with IPTG, the
morphology of the cells changed sharply , Fig. 3 C and D
show two extreme phenotypes. Although the division of
host bacteria were inhibited , the NtFtsZs∶GFPfusion pro2
teins still could be regularly distributed in the cells. This
distribution pattern was similar to the localization of GFP
tagged endogeneous FtsZ protein in E. coli[12] , and indi2
cated that NtFtsZs could recognize the potential cell divi2
sion site in E. coli and interact with the bacterial cell di2
vision proteins. On the other hand , the overexpression of
the fusion proteins in E. coli also blocked the host cell
division and resulted in a filamentous phenotype of the
cells, which is similar to the overproduction of endoge2
nous FtsZ in E. coli[ 12 ,15] . These results suggested that
NtFtsZs had the functions similar to those of bacterial.
In E. coli , FtsZ could form an obvious ring struc2
ture at the division site[12] , but in our experiments we
could not find any ringor band structures around the bac2
teria. The fusion protein was distributed mainly on the
side of cells (Fig. 3 E , arrowheads). Thus , it is unde2
fined whether NtFtsZs are involved in the formation of
prokaryotic division ring. Although the overexpression of
NtFtsZs blocked host cell division , no any ring structure
was observed. It is suggtsted that the inhibition of cell di2
vision possibly came from the right cell division site which
might be occupied by the polymerization of NtFtsZs with
endogeneous FtsZ, and finally , the formation of division
ring could be interrupted. In addition to some regular dots
polymerized along the filamentous cells , it is noteworthy
that fusion proteins also could form some special structures
(Fig. 3 , F , G, arrowheads). As above mentioned , FtsZ
is a GTPase and shares some similar structural and bio2
chemical characters with tubulin , we speculate these spe2
cial structures similar to the tubulin-like polymers formed
by polymerization of prokaryotic FtsZs in vitro or in vi2
vo[12 ,16] . These structures should be , to our knowledge ,
the first demonstration of eukaryotic FtsZ polymerization
in vivo in E. coli. However , whether these structures
represented the polymerization patterns of NtFtsZs in plas2
tids remained to be seen.
Fig. 3 . Localizations of full-length NtFtsZs∶GFP in E. coli.
A. JM109/ NtFtsZ1∶GFP cells were from colonies on plates without
IPTG. B . JM109/ GFPmut cells were from colonies on plates without
IPTG. C , D. The typical filamental cell with NtFtsZ1 ∶GFP or
NtFtsZ2∶GFP vector , showing the regularly spaced dots , respective2
ly. E. Filamentous cell showing the distribution of fusion protein.
F , G. Filamentous cells showing the speculated spiral fluores2
cences. Bars : A and B , 5μ
m ; C , D , E , F and G , 2μ
m.
2. 3 Effects of C-terminal deleted NtFtsZs on the fu2
sion protein localization in E. coli
There were two mainly localization patterns of
NtFtsZs
ΔC∶GFP in E. coli : 1)Fusion proteins was dis2
tributed at a special region , but the distribution space was
not similar to that of full-length NtFtsZs∶GFP fusion pro2
teins which had obviously regular distribution (Fig. 4 , B ,
C). 2)In addition to the fluorescence dots distributed
along bacterial cells , C-terminal deletion fusion proteins
also could be polymerized into some uncontinuous spiral
fluorescence throughout the filamentous cells (Fig. 4 , D ,
E , F , G). Compared with the localization of full-length
NtFtsZs∶GFP , the fusion proteins with C-terminal dele2
tion lost the ability to be located correctly and distributed
randomly in cells. Meanwhile , the fusion proteins also
could be polymerized in cells , suggesting that the poly2
merization function of NtFtsZs could not be affected by C-
terminal deletion. Based on the above observations , we
speculated that the C-terminal domain of NtFtsZs might be
involved in the selection of correct division site. In E.
coli , the function of FtsZ C-terminus was responsible for
the interaction of FtsZA with ZipA[14] . However , there is
no FtsZA or ZipA homolog found in higher plants till
now , thus the function of eukaryotic FtsZ C-terminal do2
main might be different from that of prokaryotic homolog.
Furthermore , it also provides a possible explain to the lo2
calization observations of full-length NtFtsZs ∶GFP in
which we could not find any ring structures , i. e. , the C-
terminal domains of NtFtsZs were incompetent for the for2
mation of prokaryotic division ring. In addition , the spiral
WANGDong et al : Localization of Two GFP-tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli 933
© 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.
fluorescence observed in C-terminal deletion experiments
further showed that the eukaryotic FtsZ also could be
polymerized into tubulin-like protofilaments as that of its
prokaryotic counterpart. This may also provides the im2
portant clues for understanding the role of FtsZ in division
of chloroplast and its morphology maintained.
Fig. 4. Localizations of C-terminal deleted NtFtsZs∶GFP in E.
coli.
A. JM109/ NtFtsZ2
ΔC∶GFP cells were from the colonies on plates
without IPTG. B , C. Filamentous cells showing the localization pat2
tern of NtFtsZ1
ΔC∶GFP or NtFtsZ2
ΔC∶GFP , respectively. D , E ,
F , G. Arrowheads showing the speculated uncontinuous , spiral
structures in filamentous cells. Bars : A , B , F and G, 10μ
m ; C ,
D and E , 5μ
m.
2. 4 Localization of N-terminal deleted NtFtsZs ∶
GFP in E. coli
The deletion of motif FTSZ-1 had no obvious effects
on the polymerization of fusion proteins , their fluores2
cence dots still could be observed in cells (Fig. 5 , A , B ,
C , D). This result indicted that the deletion of FTSZ-1
did not affect the polymerization of fusion proteins. It is
worth to note that the NtFtsZs
Δ
N1∶GFP fusion proteins
exhibited a similar localization pattern in cells as the
NtFtsZs
ΔC∶GFP did , but it remained to be seen if these
observations meant the FTSZ-1 motif was also responsible
for the function of selecting division site. The fusion pro2
teins with deletion of motif FTSZ-2 did not display any
specific localization patterns such as dots or spiral fluores2
cence. The green fluorescence could be seen throughout
the whole cells (Fig. 5 , E , F , G, H), which appears to
be the case as that in control cells. Thus , the function of
the FTSZ-2 motif of NtFtsZs is likely similar to their
Fig. 5. Localizations of N-terminal deleted NtFtsZs∶GFP in E.
coli.
A , C. Localizations of NtFtsZ1
Δ
N1∶GFP showing the irregularly
distribution of fusion proteins. B , D. Localizations of NtFtsZ2
Δ
N1∶
GFP showing the irregularly distribution of fusion proteins. Distribu2
tion patterns of NtFtsZs
Δ
N2∶GFP , E and F for NtFtsZ1
Δ
N2∶GFP ,
G and H for NtFtsZ2
Δ
N2∶GFP. Bars : A and B , 5μ
m ; C , D , E ,
F , G , H , 5μ
m.
prokaryotic homologs , that is responsible for the polymer2
ization of homogeneous/ heterogeneous FtsZ proteins. The
bright fluorescence dots at the poles of cells were thought
as inclusion bodies produced by overexpression of exoge2
neous proteins[ 12 ] , because it also could be found in con2
trol cells without NtFtsZs∶GFP plasmids (Fig. 3 , B ,ar2
rowhead). Thus , these fluorescence dots at poles might
represent a non-specific aggregation of proteins in cells.
3 Discussion
Plastids are a group of important organelles in plant
cells and involved in the whole growth and development
process of plant cells. Plastid division is an indispensible
stage for development and differentiation of plant cells.
Recent studies have revealed the role of eukaryotic FtsZ in
plastid division process. However , what patterns of FtsZ
play in controlling plastid division is still a matter of de2
bate. In this paper , we first report that the in vivo local2
ization and polymerization of eukaryotic FtsZ in E. coli ,
and provide a direct evidence to support the view that
NtFtsZs also have the typical characters of FtsZ protein in
prokaryote and can form some special structures by self-
polymerization. It was given that there were no any ring
structure formed by NtFtsZs observed in our experiments ,
whether they also act as a ring pattern in tobacco chloro2
plasts , still needs to be further studied. Moreover , all of
these results will help us to further understand which pat2
tern could represent the f unction of eukaryotic FtsZ , i . e.
it is a simple succession of prokaryotic cell division
mechinery (plastid division ring)[8 ] or a novel subcellular
structure (plastoskeleton)[7 ] , or both , in higher plants.
In addition , these results not only provide the direct evi2
dence to support the endosymbiosis hypothesis , but also
establish a feasible foundation for further studies of in vit2
ro polymerization and in vivo subcellular localization of
eukaryotic FtsZ. Certainly , there are many obvious differ2
ences exist between chloroplasts and their prokaryotic evo2
lutionary ancestors in morphology , structure of chloroplas2
ts and their situated enviroments. The experiments that
test the functions of NtFtsZs in plastid division and shape
maintenance in tobacco plants are under way.
References :
[ 1 ] Lutkenhaus J F , Addinall S G. Bacterial cell division and
the Z ring. Annu Rev Biochem , 1997 ,66 :93 - 116.
[ 2 ] Bramhill D. Bacterial cell division. Annu Rev Cell Dev
Biol , 1997 ,13 :395 - 424.
[ 3 ] Erickson H P. FtsZ , a prokaryotic homolog of tubulin ?
Cell , 1995 ,80 :367 - 370.
[ 4 ] Osteryoung KW , Vierling E. Conserved cell and organelle
division. N ature , 1995 ,376 :473 - 474.
[ 5 ] Osteryoung K W , Stokes K D , Rutherford S M , Percival A
L , Lee W Y. Chloroplast division in higher plants requires
members of two functionally divergent families with homology
to bacterial ftsZ. Plant Cell , 1998 ,10 :1991 - 2004.
[ 6 ] Strepp R , Scholz S , Kruse S , Speth V , Reski R. Plant nu2
clear gene knockout reveals a role in plastid division for the
homolog of the bacterial cell division protein FtsZ , an an2
cestral tubulin. Proc Natl Acad Sci USA , 1998 ,95 :4368 -
4373.
934 植物学报 Acta Botanica Sinica Vol. 44 No. 8 2002
© 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.
[ 7 ] Kiessling J , Kruse S , Rensing S A , Harter K , Decker E L ,
Reski R. Visualization of a cytoskeleton-like FtsZ network
in chloroplasts. J Cell Biol , 2000 ,151 :945 - 950.
[ 8 ] Vitha S , McAndrew R S , Osteryoung K W. FtsZ ring for2
mation at the chloroplast division site in plants. J Cell Biol ,
2001 ,153 :111 - 119.
[ 9 ] Miyagishima S Y , Takahara M , Kuroiwa T. Novel filaments
5 nm in diameter constitute the cytosolic ring of the plastid
division apparatus. Plant Cell , 2001 ,13 :707 - 721.
[10] Margolin W. A green light for the bacterial cytoskeleton.
Trends Microbiol , 1998 ,6:233 - 238.
[11] Sambrook J , Fritsch E F , Maniatis T. Molecular Cloning: a
Laboratory Manual. 2nd ed. New York : Cold Spring Harbor
Laboratory Press , 1989.
[12 ] 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 pro2
tein. Proc Natl Acad Sci USA , 1996 ,93 :12998 - 13003.
[13 ] RayChaudhuri D , Park J T. A point mutation converts Es2
cherichia coli FtsZ septation GTPase to an ATPase. J Biol
Chem , 1994 ,269 :22941 - 22944.
[14 ] Ma X , Margolin W. Genetic and functional analyses of the
conserved C-terminal core domain of Escherichia coli FtsZ.
J Bacteriol , 1999 ,181 :7531 - 7544.
[15 ] Ward J E , Lutkenhaus J F. Overproduction of FtsZ induces
minicell formation in E. coli.Cell , 1985 ,42 :941 - 949.
[16] Lu C , Reedy M, Erickson H P. Straight and curved confor2
mations of FtsZ are regulated by GTP hydrolysis. J
Bacteriol , 2000 ,182 :164 - 170.
烟草质体分裂蛋白 NtFtsZs 在大肠杆菌中的定位分析
王 东
1
孔冬冬
1
鞠传丽
2
胡 勇
2
何奕昆
1 ,2 3
孙敬三
13
(1. 中国科学院植物研究所 ,北京 100093 ; 2 . 首都师范大学生物系 ,北京 100037)
摘要: 分别构建了两个烟草(Nicotiana tabacum L. )质体分裂基因 Nt FtsZ1 和NtFtsZ2 与编码绿色荧光蛋白的
gfpS65A、V68L、S72A基因相融合的原核表达载体 ,并导入大肠杆菌(Escherichia coli)JM109 菌株中进行表达。全长 NtFtsZs∶
GFP 融合蛋白在菌体中有规律地定位 ,暗示 NtFtsZs 能识别大肠杆菌潜在的分裂位点 ,并能与大肠杆菌的内源 FtsZ
发生聚合作用 ;融合蛋白的诱导表达抑制了宿主菌的分裂 ,形成了明显的丝状菌体 ,证明真核生物的 ftsZ 基因与大
肠杆菌的ftsZ 基因有相似的作用 。同时构建了 NtFtsZs 不同缺失的原核表达载体 ,对这两个基因所编码蛋白不同结
构域的功能做了初步分析。实验结果表明 ,烟草 FtsZ 蛋白的 C端结构域与其在大肠杆菌细胞中的正确定位有关;
而N端结构域与 NtFtsZs∶GFP 融合蛋白的聚合有关。
关键词: 烟草 ;质体分裂基因 ;Nt FtsZ ;绿色荧光蛋白 ;缺失表达 ;原核定位
中图分类号: Q78 文献标识码 : A 文章编号 : 057727496(2002)0820931205
收稿日期 :2001207209 接收日期 :2001210219
基金项目 :国家自然科学基金(39970356);北京市自然科学基金(5992003)。
3通讯作者。E- mail : < yhe @duke. edu ; sunjs @ns. ibcas. ac. cn > 。
(责任编辑 :谢 巍)
WANGDong et al : Localization of Two GFP-tagged Tobacco Plastid Division Protein NtFtsZs in Escherichia coli 935
© 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.