JPH1036393A - New ftsz protein and dna coding for the protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new protein originating from coryne-form bacteria, having a specific amino acid sequence, participating in the formation of septum in the cell division of procaryote to control the frequency of cell division and useful as the gene of the protein for improving the breeding of industrially useful coryne-form bacteria. SOLUTION: This new Fts(Filamenting temperature sensitive) Z protein has an amino acid sequence expressed by the formula, plays an important role in the formation of septum in the cell division of procaryote and the start of the cell division and forms a ring structure by gathering to the center of a cell immediately before the formation of septum. The gene of the protein is useful for the improvement of the breeding of coryne-form bacteria to enable the control of the cell-division of industrially useful coryne-form bacteria. The FtsZ protein can be produced by producing a partial fragment of ftsZ gene by the PCR processing of chromosome DNA of Brevibacterium MJ-233, carrying out plaque hybridization using the fragment as a template and expressing a gene of a clone containing the fragment in a host.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel FtsZ protein derived from a coryneform bacterium and a DN encoding the same.
About A.

[0002]

2. Description of the Related Art fts (filamentating te)
The products of the sense sensitive genes are separated during the prokaryotic cell division by the septum (sept).
um) plays an important role in the formation and initiation thereof. The ftsZ gene, which is one of the fts gene groups, works at the early stage of septum formation, is essential for growth, and controls the frequency of cell division. In addition, it has been shown that the FtsZ protein gathers at the central site of the cell immediately before formation of the septum to form a cyclic structure [Mol. Gen. Gene
t. , Vol. 240, p. 213-220 (19
93)].

[0003] The gene encoding the FtsZ protein is described in Escherichia coli (Escherichia coli).
a. coli) [Gene, Vol. 3
6, p. 241-247 (1985)], a gene derived from Bacillus subtilis [J. Bacteriol. , Vol. 17
0, p. 4855-4864 (1988)], Rhizobium meli
loti) -derived gene [J. Bacterio
l. , Vol. 173, p. 5822-5830
(1991)], Haemophilus influenza (H
aemophilus influenzae) [Science. , Vol. 269, p.
496-512 (1995)], Drosophia melanogaster.
er) [Mol. Gen. Genet. ,
Vol. 240, p. 213-220 (199
3)] has been isolated and its base sequence has been determined.

[0004] However, there is no report on coryneform bacteria which are industrially important as far as the present inventors know.

[0005]

In general, if the origin is different, the enzyme gene is not expressed or hardly expressed in the host, so that the DNA fragment encoding the Coryneform bacterium-derived FtsZ protein that can be expressed in the Coryneform bacterium is used. Isolation was desired.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by utilizing gene recombination techniques, it has been possible to remove FtsZZ from coryneform bacteria.
The inventors have found that the protein and the DNA encoding it can be isolated, and have completed the present invention. That is, the gist of the present invention resides in the FtsZ protein represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing and the DNA encoding the same.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The FtsZ protein of the present invention and the DNA encoding the same (hereinafter abbreviated as ftsZ gene) are chromosomal DNA of coryneform bacteria, specifically, Brevibacterium flavum (Brevibac).
terium flavum) MJ-233 (FERM)
BP-1497) strain or the like, and can be determined by the method described below.

First, the coryneform bacterium, for example, Brevibacterium flavum MJ-233, is cultured according to a conventional method (for example, see Japanese Patent Application Laid-Open No. Sho 51-130592), cells are collected from the culture, and chromosomes are collected from the cells. Extract the DNA. Chromosomal DNA can be easily extracted from cells by, for example, the method described in Example 1 (A) of JP-A-5-15378.

A polymerase chain reaction (PCR) is carried out using an oligodeoxyribonucleotide reversely translated from a highly homologous portion of the primary structure (amino acid sequence) of the FtsZ protein such as Escherichia coli to obtain ft.
A partial fragment of the sZ gene was obtained. Using this fragment as a template, Southern hybridization was performed on the digested product of the chromosomal DNA restriction enzyme to determine the size of the chromosomal DNA restriction enzyme fragment containing at least a part of the ftsZ gene fragment.

Using a partial fragment of the ftsZ gene obtained by PCR as a template, λ phage containing the fragment was isolated by plaque hybridization from a gene library λFIXII. This was cloned. This chromosomal DNA fragment is cut out using an appropriate restriction enzyme, and the obtained DNA fragment is subcloned into an appropriate cloning vector, for example, pUC118 (Takara Shuzo) to transform Escherichia coli JM109 strain (Takara Shuzo). This transformant is cultured under appropriate antibiotic selection,
The cells are collected from the culture, and the plasmid is extracted from the cells by a conventional method, for example, the alkali-SDS method. By determining the base sequence of the DNA inserted into this plasmid, a DNA fragment containing the ftsZ gene of the present invention can be obtained. As a method for determining the nucleotide sequence of this DNA fragment, for example, the dideoxynucleotide enzyme method [Dide
oxy chain termination method; S
angel, F.S. et al. , Proc. N
atl. Acad. sci. U. S. A. , V
ol. 74, p. 5463, (1977)]. From the open reading frame present in the sequence of this DNA fragment, the FtsZ protein is composed of the amino acid sequence of SEQ ID NO: 1, and the gene encoding it is, for example, 1125 to 1125 in the nucleotide sequence of SEQ ID NO: 1. It is shown by the base sequence up to the th.

The ftsZ gene in the present invention is not limited to one isolated from the chromosomal DNA of a natural bacterium, for example, a coryneform bacterium, but may be a commonly used DNA synthesizer, such as a Beckman / Oligo 1000M DNA synthesizer (Oligo 1000M). DNA Syntheses
iser).

The DNA fragment of the present invention obtained from a bacterial chromosome, such as Brevibacterium flavum MJ-233, as described above, has a nucleotide sequence that is substantially the same as that of the FtsZ protein. Even if some bases are substituted with other bases, they may be deleted, new bases may be inserted, or a part of the base sequence may be transposed. Further, the base sequence may be a base sequence that hybridizes to the base sequence, and any of these derivatives is included in the DNA fragment containing the gene encoding the FtsZ protein of the present invention.

[0013]

The present invention will be described more specifically with reference to the following examples. It should be understood, however, that the examples are to be construed as helpful in obtaining a specific understanding of the invention and are not intended to limit the scope of the invention in any way. (A) Extraction of total DNA of Brevibacterium flavum MJ-233 (FERM)
BP-1497) in a semi-synthetic medium A (composition: 2 g of urea, 7 g of (NH ₄ ) ₂ SO ₄ , K ₂ HPO _{4
0.5g, KH 2 PO 4 0.5g,} MgSO 4 · 7H 2
_{O 0.5g, FeSO 4 · 7H 2} O 6mg, MnSO
₄ · _{4~6H 2} O 6mg, yeast extract 2.5g, casamino acid 5g, biotin 200 [mu] g, thiamine hydrochloride
200 μg and 20 g of glucose are dissolved in distilled water to obtain 1
The cells were collected after culturing in 1 liter until the late logarithmic growth phase.

The obtained cells were lysozyme at 10 mg / m
1 solution [composition: 10 mM NaCl,
20 mM Tris buffer (pH 8.0), 1 mM ED
TA • 2Na] in 15 ml. To the suspension was added proteinase K at a final concentration of 100 μg / ml,
Incubated for 1 hour at 7 ° C. Next, sodium dodecyl sulfate was added to a final concentration of 0.5%,
The cells were lysed by incubation at 50 ° C. for 6 hours. An equal amount of a phenol / chloroform solution was added to the obtained lysate, and the mixture was gently shaken at room temperature for 10 minutes.
The mixture was centrifuged at 5,000 xg for 20 minutes at 0 to 12 ° C, and the supernatant fraction was collected. Sodium acetate was added to the supernatant fraction to a concentration of 0.3 M, and then twice the amount of ethanol was gently added. Capture the DNA between the aqueous and ethanol layers with a glass rod,
This was washed with 70% ethanol and air-dried. The obtained DNA was added to a solution [composition: 10 mM Tris buffer (pH
7.5) 5 ml of 1 mM EDTA · 2Na] was added, and the mixture was allowed to stand at 4 ° C. overnight, and then subjected to an experiment.

(B) Collection of a partial fragment of the ftsZ gene Escherichia col
i) -derived gene [Gene, Vol. 36, p.
241-247 (1985)], a gene derived from Bacillus subtilis [J. Bacteriol. , Vol. 170,
p. 4855-4864 (1988)], Rhizobium melilot.
i) derived genes [J. Bacteriol. , V
ol. 173, p. 5822-5830 (199
1)], Drosophila Melanogaster (Drosop)
Himelanogaster) [Mo
l. Gen. Genet. , Vol. 240, p. 2
13-220 (1993)], a PCR primer DNA for gene cloning based on the sequence of the homologous portion of the amino acid sequence estimated based on the base sequence of the ftsZ gene of
Was designed. Primer 1: GGNGTNGGNG GNGGNGGNGA YAAYGCNGT (SEQ ID NO: 2) Primer 2: GGNGCNGCNC CNGTNCCNGC (SEQ ID NO: 3) (N indicates (AGTC), Y indicates (TC).)

Using the primers and the chromosomal DNA isolated in (A) as a template, a reaction mixture 10
The polymerase chain reaction was performed using 0 μl. (The concentration represents the final concentration.) 25 units / ml Taq DNA polymerase 10 mM Tris-HCl buffer (pH 8.
0) 50 mM KCl 1.5 mM MgCl ₂ 0.25 mM dATP 0.25 mM dCTP 0.25 mM dGTP 0.25 mM dTTP 0.5 μg / ml Chromosomal DNA 1 μM Primer DNA 1 (SEQ ID NO: 2; amino acid sequence 15 to 24 described in SEQ ID NO: 1) 1 μM primer DNA2 (SEQ ID NO: 3, corresponding to amino acid sequences 106 to 112 described in SEQ ID NO: 1)

The reaction conditions of the polymerase chain reaction are, for example, 25 cycles of 94 ° C., 1 minute, 55 ° C., 2 minutes, 72 ° C., 3 minutes. Then, the DNA fragment amplified by the above reaction is converted into plasmid p according to the following method.
It was cloned into GEM-T. That is, the final concentrations were 50 mM Tris-HCl buffer (pH 7.9),
10 mM MgCl ₂ , 20 mM dithiothreitol, 1 mM ATP, 100 unit / ml T4DN
A ligase, 5 μg / ml pGEM-T vector, 1
Each component was added to give a reaction mixture of 10 μl as a μg / ml PCR product, and reacted at 16 ° C. for 3 hours to bind the PCR product DNA. Then, the conventional method [J. Mol. Biol. , 53, 159 (1970).
According to E. coli.
E. coli JM109 was transformed. The obtained transformant was added to a selective medium [composition: 10 g of tryptone, yeast extract].
5 g, NaCl 5 g, agar 15 g, ampicillin 5
0 mg, isopropiothiogalactoside 0.238
g, X-gal 0.2 g, dimethylformamide 2
was dissolved in distilled water to make 1 liter] and cultured at 37 ° C. for 16 hours. The colonies thus obtained were subjected to blue / white color screening. The white colonies grown on the selection medium were picked up to a final concentration of 5 ampicillin.
An L culture solution containing 10 μg / ml (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl dissolved in distilled water to make 1 liter) was inoculated and cultured at 37 ° C. for 7 hours. The culture was centrifuged at 8,000 × g for 10 minutes at 4 ° C. to collect the cells. Alkali-
SDS method [T. Maniatis, E .; F. Frit
sch, J.A. Sambrook, Molecula
r cloning, p. 90-91 (1982)].

Next, the nucleotide sequence of the chromosomal DNA fragment inserted into the obtained plasmid was determined by the dideoxynucleotide enzyme method. Specifically, the plasmid DNA extracted from the above culture was used for Catalist 800 Molecular Biology Laboratory Station (CATALYST 800 Molecular) manufactured by Perkin Elmer.
r Biology Labostation; Pe
The reaction was carried out using Rkin-Elmer according to the protocol, and the base sequence of the inserted DNA fragment of the plasmid was determined using a 373A DNA sequencer manufactured by Perkin-Elmer.

By comparing the homology between the protein obtained by translating the determined nucleotide sequence and the known FtsZ protein of Escherichia coli, it was found that a part of the ftsZ gene of Brevibacterium flavum MJ-233 ( 43 to 72 in the base sequence described in SEQ ID NO: 1). (C) Determination of size of chromosomal DNA restriction enzyme fragment containing partial fragment of ftsZ gene Chromosomal DNA was converted to restriction enzymes BamHI, EcoRI, Hi
ncII, HindIII, KpnI, PstI, Pv
It was digested with uII and SalI, respectively. These are Onc
A nylon membrane filter for Southern hybridization was prepared using Probe Tech 2 manufactured by Or Corporation.

Using the partial fragment of the ftsZ gene obtained by PCR as a template and a label
³² P] using dCTP AA0005, Takara Shuzo Co., Ltd. R
andom Primer DNA Labellin
g Kit Ver. Probes were labeled in two ways.
A filter having the following composition [5 × SSC solution, 5 ×
Denhardt's solution, 0.5% sodium dodecyl sulfate,
SIGMA SALMON TESTES DNA f
or Hybridization (10mg / m
1) 0.1 mg / ml] at 65 ° C for 2 hours. The 20 × SSC solution is
The following composition [3M NaCl, 0.3M sodium citrate], 100 × Denhardt's solution has the following composition [bovine serum albumin 2%, polyvinylpyrrolidone 2%, ficoll 2%].

The probe prepared above was added, and Southern hybridization was carried out at 65 ° C. overnight. The filter was washed with 2 × SSC, 0.1% SDS at 65 ° C. for 15 minutes with gentle shaking. Next, filter 1
The plate was washed with × SSC and 0.1% SDS at 65 ° C. for 15 minutes with gentle shaking. After air drying the filter,
Autoradiography was performed. The reading is Bio Imaging Analyzer BA manufactured by Fuji Photo Film Co., Ltd.
S-2000 was used.

As a result, the size of the chromosomal DNA restriction enzyme fragment including the partial fragment of the ftsZ gene was as follows: BamHI fragment, EcoRI fragment, HincII fragment, HindII fragment.
The I fragment, KpnI fragment, PstI fragment, PvuII fragment, and SalI fragment were about 1.4 kb and 4.1 k, respectively.
b, 0.8b, 4.2 kb, 2.7 kb, 1.7 kb,
They were 1.9 kb and 8.0 kb.

(D) EcoRID containing ftsZ gene
Isolation of NA fragment (4.1 kb) Escherichia coli P2329 was inoculated into an LB culture solution supplemented with 0.2% maltose and 10 mM MgSO ₄ and cultured at 37 ° C. And the gene library λF
The P2329 culture solution was mixed with 400 μl of the IXII phage solution and cultured at 37 ° C. for 15 minutes. Next, 4 ml of λ top agar (preserved at 50 ° C.) was added, spread uniformly on a λ plate, and cultured at 37 ° C. overnight.

The nitrocellulose filter was gently placed on the λ plate so that air did not enter, and the mark points previously written on the filter were transferred to the plate. The filter was peeled off and the adsorption surface was placed on a filter paper immersed in the following solution and treated sequentially for 5 minutes ([0.5M NaOH,
1.5M NaCl], [1M Tris-HCl (pH
7.5), 0.75 M NaCl], 2 x SSC). After drying the filter, it was heated at 80 ° C. for 30 minutes to immobilize DNA on the filter.

The filter was mixed with the following solution [5 × SSPE,
1x Denhardt's solution, 50% formamide, SIGMA
SALMON TESTES DNA For H
ybridization (10 mg / ml)
[1 mg / ml] at 42 ° C for 1 hour. The composition of 20 × SSPE is 3.6M
NaCl, 0.2 M NaH ₂ PO ₄ , 0.02 M E
DTA. Next, the DNA probe prepared in the above (C) was added, and hybridization was carried out at 42 ° C. overnight.

The filter was washed with 5 × SSPE, 1 × Denhardt's solution, 50% formamide solution at 42 ° C. for 15 minutes with gentle shaking. Then filter 2x
SSPE, 42% with 0.1% sodium dodecyl sulfate solution
Washing was carried out with gentle shaking at 15 ° C. for 15 minutes. After air-drying the filter, autoradiography was performed.
For reading, a bioimaging analyzer BAS-2000 manufactured by Fuji Photo Film Co., Ltd. was used.

The soft agarose of the target plaque was crushed and suspended in 200 μl of SM buffer. The above solution 10
μl was mixed with 300 μl of Escherichia coli P2329 strain cultured at 37 ° C. for 3 to 4 hours, top agarose was added thereto, and the mixture was spread on a λ plate. The cells were cultured overnight at 37 ° C. to form plaques. Add 4 ml of SM buffer to the λ plate, scrape off the top agarose,
Shake gently for hours. The supernatant was transferred to a new tube so that top agarose was not mixed, and a few drops of chloroform were added. Then, the mixture was centrifuged at 5,000 rpm for 5 minutes to obtain a supernatant. Further, DNase and RNase (final concentration 1 μg / ml) were added, and the mixture was kept at 37 ° C. for 15 minutes. An equivalent amount of 20% polyethylene glycol (average molecular weight: 6,000) -2M NaCl was added, the mixture was allowed to stand on ice for 1 hour, and then centrifuged at 10,000 rpm at 4 ° C. for 10 minutes, and the supernatant was completely removed. 250 μl of Tris-E
DTA buffer was added to suspend, 5 μl of 10% sodium dodecyl sulfate was added, and the mixture was heated at 68 ° C. for 5 minutes.
One liter of 5M NaCl was added, an equal amount of phenol / chloroform was added, and the mixture was suspended well. 12,000 rpm, 1
After centrifugation for 0 minutes, the aqueous layer was transferred to a new tube. After isopropanol precipitation, 70% ethanol washing and drying,
Suspended in 50 μl of Tris-HCl buffer.

ΛFIXII DNA obtained by the above operation
Was cut with EcoRI. The digest was subjected to agarose gel electrophoresis to obtain about 4.1 kb of EcoRI containing the ftsZ gene.
Was separated and purified. This EcoRI fragment was subcloned into pBluescript SK-. 4.1 kb D of subcloned EcoRI
PBluescript SK- containing the NA fragment
It was named S-ftsZ.

(E) Determination of the nucleotide sequence of the ftsZ gene The nucleotide sequence of the approximately 4.1 kb EcoRI DNA fragment contained in pBS-ftsZ obtained in (D) was determined as follows:
Dideoxynucleotide enzymatic method [Dideoxy ch
ain termination method; Sanger,
F. et al. , Proc. Natl. Ac
ad. sci. U. S. A. , Vol. 74,
p. 5463, (1977)].

Ligation of these individual sequences was performed using a sequence analysis software Autoassembler (Autoassembler) manufactured by Perkin-Elmer. As a result, the 4.1 kb EcoRI was used.
DNA fragments of the known Escherichia coli Fts
A protein with high homology to Z protein (homology 4
7%) (open reading frame described in SEQ ID NO: 1). That is, by comparing homology between the amino acid primary structure of the protein described in Sequence 1 and the amino acid primary structure of the known FtsZ protein of Escherichia coli, the 4.1 kb EcoRI DNA fragment was found to be Brevibacterium flavum MJ- It was found to contain 233 ftsZ genes.

[0031]

Industrial Applicability By breeding and improving coryneform bacteria using the DNA fragment containing the ftsZ gene provided by the present invention, it is possible to control the cell division of coryneform bacteria which is industrially useful.

[0032]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1125 Number of chains: double-stranded Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin: Organism: Brevibacterium flavum (Brevibacteri)
um flavum) Strain name: MJ-233 Sequence characteristics: Symbol indicating characteristics: CDS Location: 1-1122 Method for determining characteristics: Sequence ATG ACC TCA CCG AAC AAC TAC TCG CCA AGA TTG GAG CTC GTC GGC GTG 48 Met Thr Ser Pro Asn Asn Tyr Ser Pro Arg Leu Glu Leu Val Gly Val 1 5 10 15 GGC GGC GGC GGA GAT AAT GCT GTC AAC CGC ATG ATT GAA GAA GGC CTC 96 Gly Gly Gly Gly Asp Asn Ala Val Asn Arg Met Ile Glu Glu Gly Leu 20 25 30 AAA GGC GTG GAG TTC ATC GCG GTG AAC ACC GAC TCG CAG GCT CTC ATG 144 Lys Gly Val Glu Phe Ile Ala Val Asn Thr Asp Ser Gln Ala Leu Met 35 40 45 TTC TCT GAT GCC GAC GTA AAG CTC GAT ATC GGA CGT GAA GCT ACC CGT 192 Phe Ser Asp Ala Asp Val Lys Leu Asp Ile Gly Arg Glu Ala Thr Arg 50 55 60 GGT CTT GGT GCC GGC GCG AAC CCA GAA GTT GGA CGT GCC TCG GCA GAG 240 Gly Leu Gly Ala Gly Ala Asn Pro Glu Val Gly Arg Ala Ser Ala Glu 65 70 75 80 TAT CAC AAG AAC GAA ATC GAA GAA ACC ATC AAG GGC GCC GAC ATG GTC 288 Tyr His Lys Asn Glu Ile Glu Glu Thr Ile Lys Gly Ala Asp Me t Val 85 90 95 TTC GTT ACC GCC GGC GAA GGT GGT GGg gCG GGT ACA GGA GCt GCa CCA 336 Phe Val Thr Ala Gly Glu Gly Gly Gly Ala Gly Thr Gly Ala Ala Pro 100 105 110 GTC GTG GGA CGG ATC GCC AAG AAG ATG GGC GCA CTG ACC ATT GGT GTT 384 Val Val Gly Arg Ile Ala Lys Lys Met Gly Ala Leu Thr Ile Gly Val 115 120 125 GTG ACC AAG CCT TTC GAG TTC GAA GGC CGT CGC CGT ACT CGC CAG GCA 432 Val Thr Lys Pro Phe Glu Phe Glu Gly Arg Arg Arg Thr Arg Gln Ala 130 135 140 GAA gAA GGC ATC GCA GCA CTG AAG GAG GTC TGC GAC ACT CTC ATC GTT 480 Glu Glu Gly Ile Ala Ala Leu Lys Glu Val Cys Asp Thr Leu Ile Val 145 150 155 160 ATT CCA AAC GAC CGC CTG CTT GAG CTG GGC GAT GCT AAC CTG TCC ATC 528 Ile Pro Asn Asp Arg Leu Leu Glu Leu Gly Asp Ala Asn Leu Ser Ile 165 170 175 ATG GAA GCG TTC CGC GCA GCC GAT GAA GTT CTC CAC AAT GGT GTT CAG 576 Met Glu Ala Phe Arg Ala Ala Asp Glu Val Leu His Asn Gly Val Gln 180 185 190 GGT ATT ACC AAC CTG ATC ACC ATC CCT TGT ATC AAC GTG GAC TTC GCG 624 Gly Ile Thr Asn Leu Ile Thr Ile Pro Cys Ile Asn V al Asp Phe Ala 195 200 205 GAC GTT CGC TCC GTC ATG TCC GAA GCT GGT TCC GCA CTC ATG GGT GTG 672 Asp Val Arg Ser Val Met Ser Glu Ala Gly Ser Ala Leu Met Gly Val 210 215 220 GGC TCT GCA CGT GGG GAC AAC CGC GTT GTC TCT GCA ACC GAG CAG GCC 720 Gly Ser Ala Arg Gly Asp Asn Arg Val Val Ser Ala Thr Glu Gln Ala 225 230 235 240 ATC AAC TCT CCA CTT CTC GAA GCA ACC ATG GAC GGC GCA ACT GGC GTC 768 Ile Asn Ser Pro Leu Leu Glu Ala Thr Met Asp Gly Ala Thr Gly Val 245 250 255 CTG CTG TCC TTT GCT GGT GGA TCC GAC CTG GGC CTC ATG GAA TCA ACG 816 Leu Leu Ser Phe Ala Gly Gly Ser Asp Leu Gly Leu Met Glu Ser Thr 260 265 270 CAG CTG CAT CCA TGG TCC GTG AGC GTT CCG ATG AAA TTC aAC CTC ATC 864 Gln Leu His Pro Trp Ser Val Ser Val Pro Met Lys Phe Asn Leu Ile 275 280 285 TTC GGT ACC ATC ATC GAC GAC AAC CTG GGC GAC GAA GTC CGC GTA ACC 912 Phe Gly Thr Ile Ile Asp Asp Asn Leu Gly Asp Glu Val Arg Val Thr 290 295 300 GTC ATC GCG ACC GGT TTT GAC GCA GCT CGC GCA AGC GCC GCT GAG AAC 960 Val Ile Ala Thr Gly Phe Asp Ala Ala A rg Ala Ser Ala Ala Glu Asn 305 310 315 320 CGC CGC GCA GGC ATC CCA GCT GCA CCT GCA GCT GAG CCA GTC CAG CAG 1008 Arg Arg Ala Gly Ile Pro Ala Ala Pro Ala Ala Glu Pro Val Gln Gln 325 330 335 CAG CAt GTC CCA ACC ACC AAC GCA ACC TTC CAC CAG AGA AGA AGC ATC 1056 Gln His Val Pro Thr Thr Asn Ala Thr Phe His Gln Arg Arg Ser Ile 340 345 350 TTC GGT GGT GCA CGT GAg GAG AAC GtA TCC TTA CCT GTC CCG CTC TGC 1104 Phe Gly Gly Ala Arg Glu Glu Asn Val Ser Leu Pro Val Pro Leu Cys 355 360 365 TGG TGC ACG TCA TCG CAT TGA 1125 Trp Cys Thr Ser Ser His 370

SEQ ID NO: 2 Sequence length: 29 Number of strands: single-stranded Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GGNGTNGGNG GNGGNGGNGA YAAYGCNGT

SEQ ID NO: 3 Sequence length: 20 Number of chains: single-stranded Sequence type: nucleic acid Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GGNGCNGCNC CNGTNCCNGC

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location // (C12N 1/21 C12R 1:19) (C12N 15/09 ZNA C12R 1:13) (72 ) Inventor Kazuhisa Hatakeyama 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Prefecture Inside the Tsukuba Research Laboratory, Mitsubishi Chemical Corporation (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Mitsubishi Tsukuba In the laboratory

Claims (3)

[Claims] 1. An FtsZ protein represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A gene encoding the FtsZ protein according to claim 1. 3. The nucleotide sequence of SEQ ID NO: 1 in the sequence listing
A gene encoding the FtsZ protein represented by the 1122nd to 1122nd nucleotide sequences.