JP3484954B2 - DNA gyrase inhibitor protein - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein (DGI) which inhibits bacterial DNA gyrase activity and a method for producing the same. Further, the present invention relates to a method for screening and identifying an antibacterial drug or the like by assaying the effect of a compound on the activity or expression of DGI. In addition, antisense DNA (or RN) of the gene encoding DGI
A) and antibodies to DGI.

[0002]

2. Description of the Related Art Since the discovery of penicillin, various antibiotics have been used for treating bacterial infections and have made a great contribution to medical treatment. The main antibiotics currently in clinical use are β
-Lactam antibiotics and new quinolone antibiotics. The mechanism of action of new quinolone antibiotics is the bacterial D
It is known to be an obstacle to NA gyrase.

Bacterial DNA gyrase is chromosomal DN
It is an enzyme essential for bacterial replication and growth, such as introducing a negative superhelical structure into A and unwinding and separating the entangled daughter DNA immediately after replication. It is composed of two subunits (A and B). It is configured.

Compounds such as novobiocin and cyclothiazine other than new quinolone antibiotics are known as DNA gyrase inhibitors, but no proteinaceous inhibitors are known.

Regarding the regulation of DNA gyrase activity,
Horiuchi et al. Reported that the LetD protein encoded on the F factor, which is a plasmid of Escherichia coli, is a DNA gyrase.
The LetA protein was used to inactivate the gyre.
It has been reported that they are involved in the reactivation of the moss (J
individual of Biological Chem
Istly, Vol. 267, 12244-12251,
1992). However, inactivation by the LetD protein was observed only in experiments using whole cells, and in vitro
No inactivating effect was observed in the reconstitution experiment in.
In addition, a DNA gyre encoded on the bacterial chromosome
There is no report on the inhibitory protein.

Recently, the emergence of resistant bacteria to conventional β-lactam and new quinolone antibiotics has become a serious problem, and antibiotics with a new mechanism of action which are effective against these resistant bacteria are desired. There is.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a DNA gyrase inhibitory protein (DGI) and a method for producing the same, which are useful for the creation of a new mechanism of action of antibiotics. Further, it is to provide a method for screening and identifying an antibacterial drug or the like having a new mechanism of action that modulates the activity of DGI or its expression using DGI or its gene.

[0008]

[Means for Solving the Problems] The present inventors have developed Escherichia coli DNA using novobiocin-affinity column chromatography.
In the process of purifying gyrase, the supercoiling activity of each fraction was measured and analyzed by SDS-polyacrylamide electrophoresis.
It was found that there were fractions that did not show supercoiling activity even though they contained the holoenzyme of A gyrase, and these fractions commonly contained a protein of about 18 kDa. The inventors
The completely new finding that the protein of kDa has an action of inhibiting the activity of DNA gyrase (supercoiling activity) was found, and this 18 kDa protein was named DNA gyrase inhibitor protein (DGI) (also referred to as GyrI). The inventors have further cloned a gene (dgi gene) (also called gyrI gene) encoding this protein from the chromosome of E. coli and
Established the manufacturing method. In addition, a system for measuring DGI activity using purified DGI and a system for measuring DGI expression using the promoter region of the dgi gene were constructed. Furthermore, by modulating the activity or expression of DGI,
The present invention has been completed by finding that the growth of bacteria can be suppressed.

That is, the present invention is a protein (DNA gyrase inhibitory protein; DGI) which inhibits bacterial DNA gyrase activity. In addition, D that encodes DGI
Host cell transformed with a replicable expression vector containing NA, and DG comprising culturing this host cell
It is a manufacturing method of I. Furthermore, the DNA of DGI
A method for identifying a pharmaceutical compound, which comprises assaying the effect of modulating a gyrase inhibitory activity, and a method for identifying a pharmaceutical compound, which comprises assaying the effect of modulating the expression of a gene encoding DGI. . Also, DG
It is an antibody that recognizes antisense DNA or antisense RNA of a gene encoding I and DGI.

E. coli-derived D is shown in SEQ ID NO: 1 of the sequence listing below.
The N-terminal (16 residues) amino acid sequence of GI is shown in SEQ ID NO: 2 and SEQ ID NO: 3 as dgi encoding E. coli DGI.
The nucleotide sequences of synthetic primer DNAs used for gene cloning are shown in SEQ ID NO: 4 and SEQ ID NO: 5 as Escherichia coli DG.
The base sequence of the synthetic primer DNA used for the preparation of the I expression vector is shown in SEQ ID NO: 6, and the base sequence of the DNA fragment containing the E. coli dgi gene and the DGI encoded therein.
The amino acid sequence of Escherichia coli DGI is shown in SEQ ID NO: 7, respectively. SEQ ID NO: 8 shows the nucleotide sequence of a DNA fragment containing the dgi gene derived from Shigella genus microorganism and the amino acid sequence of DGI encoded therein, and SEQ ID NO: 9 shows the amino acid sequence of the DGI derived from Shigella genus microorganism, respectively. It was

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION As the DGI-producing bacterium of the present invention, Escherichia coli can be preferably used. Specific strains include E
scherichia coli KL16 strain (National Institute of Genetics, Center for Preservation of Organisms for Genetic Experiments, Acce
session No. ME8002), K-12 strain (Genetics 38, 51-64, 19).
53), the same ML1410 (Microbilog
y and Immunology Volume 22, 36
7-375, 1978), ATCC 2592.
2, the same NIHJ JC-2, the same JM109 strain (AT
CC 53323), GI 724 (Invitro
gen, USA) and the like.

The DGI of the present invention is widely and universally present in various bacteria other than Escherichia microorganisms such as Escherichia coli. Examples of such bacteria include Shigella genus and Citrobacter. Examples thereof include microorganisms belonging to the genera, Pseudomonas, Bacillus, Enterococcus, Staphylococcus. Examples of specific strains include Shigella boydi IID627 (N
IHJ 1130 Type7) (Japanese Journal of Bacteriology No. 50)
Volume 1019-1031, 1995, Table 1-1), ATCC3
5964, ATCC49348, Shigella dysenteriae (Shigella dysenteriae)
IID633 (NIHJ 177249 Type3)
(Japanese Journal of Bacteriology, Volume 50, pp. 1019-1031, 1995, Table 1
-1), the same ATCC13313, the same ATCC2335
1, ATCC49345, Shigera Sonei (Shi
gella sonnei) TRRL10805, same
ATCC 11060, ATCC 29930, Citrobacterium Freundy
reundii) IID976 (NIH 17), Pseudomonas aeruginosa (Pseudomonas)
aeruginosa) ATCC27853, Bacillus subtilis
ATCC6633, Enterococcus faecalis AT
CC29212, Staphylococcus aureus (S
taphylococcus aureus) RN45
0 (Journal of Bacterology
(Vol. 174, pp. 4952-4959, 1992)
And so on.

DGI is purified by combining various purification methods such as ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, and gel filtration chromatography from bacterial cultures to determine its physiological activity (DNA gyrase supercoiling activity). Can be carried out, for example, as follows.

When utilizing the property that DGI exists in association with DNA gyrase, for example, a method for purifying DNA gyrase using a bacterial cell extract in the logarithmic growth phase (A
Oyama et al., Antimicrobial Agen
ts and Chemotherapy, Volume 32,
104-109, 1988), nucleic acid removal,
Ammonium sulfate precipitation and fractionation using a novobiocin-affinity column are performed. Novobiocin-affinity chromatography is used for fractional purification of DNA gyrase because novobiocin binds to DNA gyrase. For each fraction, the supercoiling activity of DNA gyrase was measured, and SDS-polyacrylamide gel electrophoresis was carried out to confirm the presence of a protein corresponding to the DNA gyrase holoenzyme by SDS-polyacrylamide gel electrophoresis. Nevertheless, a fraction showing no supercoiling activity, that is, a fraction in which DGI is mixed with DNA gyrase is collected. DGI can be isolated and purified by subjecting this fraction to gel filtration using Sephadex G-75.

Alternatively, a bacterial cell extract in the logarithmic growth phase is used, ammonium sulfate precipitation, strong anion exchange chromatography using Q-Sepharose Fast Flow column (Pharmacia), and TSKgel Toyopearl HW-55 column (Tosoh) are used. DGI can also be purified by gel filtration and SDS polyacrylamide electrophoresis.

The supercoiling activity of DNA gyrase is determined, for example, by Antimicrobial Agen.
ts and Chemotherapy, Volume 32,
It can be measured according to the method described on pages 104-109, 1988. The DGI activity, that is, the action of inhibiting the supercoiling activity of DNA gyrase can be measured using a supercoiling activity measurement system.

Purified DGI derived from E. coli has a molecular weight of about 18
It was a protein of kDa. The N-terminal amino acid sequence is shown in SEQ ID NO: 1 below.

Nucleic acid sequence data bank EMBL / Genb
A homology search using ank / DDBJ revealed that the N-terminal sequence of DGI was Escherichia coli K-12 strain chromosome D.
SbcB region of NA (EMBL registration number U00009)
It coincided with the N-terminal amino acid sequence of the hypothetical protein YeeB. The molecular weight of DGI was also in agreement with that of the hypothetical product YeB. From these facts, the inventors have found that YeB is DGI,
The gene encoding I (dgi gene) was considered to be in the portion containing the translation region of YeeB, and the Escherichia coli dgi gene was cloned based on the nucleotide sequence before and after the translation region of the YeeB gene.

EMBL accession number U00009 describes the nucleotide sequence of the sbcB region of Escherichia coli K-12 strain chromosomal DNA, and describes the primary amino acid sequence of the structural gene product hypothesized by the registrants. ing. However, with regard to such virtual products (YeeB etc.), there is nothing known about the most important information, physiological activity or function, and there is no suggestion, and whether or not they are actually expressed in cells. Not even known. In addition, the sbmC gene (gene product SbmC) in the sbcB region, which has the same sequence as that of yeB.
Have been reported (EMBL Accession No. X84885, and Molecular Microbiology 18: 301-311, 1).
995). However, regarding the sbmC gene, the association with resistance to the peptide antibiotic Microcin B17, and
Although there is a description that it is presumed to be one of the SOS genes, what kind of physiological activity and function the gene product has has not been clarified.

Cloning of the Escherichia coli dgi gene was carried out using y in the sbcB region (EMBL accession number U00009).
Using a primer designed and synthesized based on the nucleotide sequences around the eeB translation region, E. coli chromosomal DNA was used as a template for the polymerase chain reaction (PCR).
It can be cloned by carrying out a hain reaction). The PCR product may be cleaved with an appropriate restriction enzyme, if necessary, and then ligated to a vector plasmid that can be replicated in an E. coli host.

The dgi gene derived from Escherichia coli or a bacterium other than E. coli is, for example, a chromosomal DN of the target bacterium.
It can be easily cloned by preparing an A library and screening using a labeled DNA fragment containing a part or all of the Escherichia coli dgi gene as a probe. For example, a DNA fragment of the dgi gene consisting of the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 8 may be used as a probe to select a gene that hybridizes with this fragment under stringent conditions. The DNA sequence of the dgi gene can be determined by determining the DNA base sequence of the obtained positive clone, and the amino acid sequence of DGI can be obtained from its translation region. Such a dgi gene derived from a bacterium other than Escherichia coli generally has 70% or more, preferably 80% or more, and more preferably 90% or more homology in the nucleotide sequence with the dgi gene of Escherichia coli.

The DNA library is, for example, "Mol.
electrical Cloning "(Sambrook, J., Fri
tsch, EF and Maniatis, T., Cold Spring Harbor
Published by Laboratory Press in 1989). Alternatively, if a commercially available library is available, this may be used.

D encoding DGI such as dgi gene
NA can be used to express DGI in host cells by recombinant techniques. DGI-encoding DNA is a suitable promoter (for example, when E. coli is used as a host, λpL promoter, trp promoter,
a vector that is connected to the downstream of the lac promoter, T7 promoter, etc., and functions in the host microorganism (for example, when E. coli is used as the host, pBR322, pUC18,
pUC19) to construct an expression plasmid.
Alternatively, the DGI-encoding DNA may be ligated to an expression vector containing an appropriate promoter. DG
When a large amount of I is expressed, normal growth of the host cell is inhibited. Therefore, for the purpose of large-scale expression of DGI, it is preferable to use a vector containing a promoter capable of controlling the expression induction. Preferably, such an expression vector is p
LEX (manufactured by Invitrogen), pET (manufactured by Novagen) and the like can be mentioned.

The resulting expression plasmid (ie DG
(Reproducible expression vector containing DNA encoding I)
By culturing the host cell transformed with, the desired DGI can be obtained from the obtained culture solution or cells.

As the DGI-encoding DNA, a naturally occurring gene of a microorganism may be used, but it is not limited thereto. For example, in the case of E. coli DGI,
DNA encoding the amino acid sequence represented by SEQ ID NO: 7
In the case of DGI of a microorganism belonging to the genus Shigella, it may be any DNA that encodes the amino acid sequence represented by SEQ ID NO: 9. Since the codons corresponding to amino acids are known,
Without being limited to the native dgi gene, a DNA corresponding to the amino acid sequence can be designed and used as a DNA encoding a polypeptide. One to six types of codons encoding one amino acid are known, and the choice of codons is arbitrary. For example, a sequence with higher expression efficiency is designed in consideration of the frequency of codon usage of the host used for expression. can do. The DNA having the designed nucleotide sequence can be obtained by chemical synthesis, partial modification of naturally occurring gene, or the like. Partial modification of artificial nucleotide sequence, mutation introduction, using a synthetic oligonucleotide encoding the desired modification as a primer, known site-specific mutagenesis method (Mark, DF et al., Proceedings of Na
National Academy of Sciences, Vol. 81, pages 5662-5666 (1984)) and the like.

In Escherichia coli in which a large amount of DGI is expressed, abnormal cell division is observed. Further, when the antisense RNA of the dgi gene is expressed to suppress the expression of DGI, elongation of the bacterium is observed, and such morphological abnormality is
It is also observed when Escherichia coli is treated with a new quinolone drug that is a gyrase inhibitor (Takeshi Nishino et al., Chemo
therapy. , 41 (s-5), 50-66, 19
93). From these, it is considered that a pharmaceutical compound that modulates the activity of DGI (enhances or inhibits) or modulates the expression of dgi gene (enhances or suppresses) is useful as an antibacterial agent.

By using the DGI of the present invention, it can be assayed whether the test compound has an action of modulating the activity of DGI (DNA gyrase inhibitory activity). Further, by using the dgi gene promoter of the present invention, it is possible to test whether or not the test compound has an action of modulating the expression of the dgi gene. With such an assay, for example, pharmaceutical compounds useful as antibacterial agents can be screened and identified.

[0028] As a method for efficiently assaying whether a test compound has an action of modulating the activity of DGI using the promoter of the dgi gene of the present invention, for example, an indicator is provided downstream of the promoter region in the dgi gene. Examples include a method of linking genes and using a recombinant plasmid designed to express an indicator gene under the control of a dgi gene promoter. A host cell can be transformed with the obtained recombinant plasmid, and the promoter activity can be efficiently measured using the expression of the indicator protein in the transformed host cell as an index. Since there is a problem that the expression efficiency of the promoter is different between different types, it is desirable that the origin of the dgi promoter is the same as that of the microorganism used as the host.
Examples of the indicator protein (indicator gene) include β-galactosidase (lacZ gene), luciferase (luc gene), alkaline phosphatase (phoA gene), and the like.

Examples of substances that modulate the expression of the dgi gene include antisense DNA or RNA of the dgi gene. Such DNA or RNA can be obtained by, for example, chemical synthesis. In addition, antisense RNA can be obtained by expressing in a host a plasmid in which a fragment containing a part of a gene is inserted into an expression vector in the reverse direction (in the antisense direction).

Further, the purified DGI can be used to obtain an antibody (monoclonal or polyclonal antibody) that specifically recognizes DGI. For example, a polyclonal antibody can be prepared by inoculating an appropriate host animal (eg, rabbit or mouse) with purified DGI, a fragment thereof, a synthetic peptide having a partial sequence thereof, or the like, and collecting an antiserum by a usual method. Can be manufactured. A monoclonal antibody can be produced by a technique such as a usual hybridoma method using purified DGI, a fragment thereof, or a synthetic peptide having a partial sequence thereof as an antigen. The obtained antibody can be used for detection, quantification, purification, etc. of DGI.

The DGI of the present invention also includes a fragment thereof or a homologue thereof. Such a fragment or homologue may have the same biological activity as DGI (substantially the same species and efficacy), or may have immunological equivalence. Specifically, for example, in addition to the DGI having the amino acid sequence shown in SEQ ID NO: 7 or 9, for example, one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 7 or 9. Alternatively, those having an added amino acid sequence can be mentioned. Amino acid deletions, substitutions or additions may be carried out as long as the ability to inhibit the DNA gyrase activity is not lost, and are usually 1 to about 30, preferably 1 to about 15, more preferably 1 to about 7. It is an individual.
Such a protein usually has an amino acid sequence homology of 80% or more, preferably 90% or more, more preferably 95% or more with the amino acid sequence shown in SEQ ID NO: 7 or 9.

The gene encoding DGI of the present invention includes DNA having the nucleotide sequence shown in SEQ ID NO: 6 or 8 and DNA having the nucleotide sequence shown in SEQ ID NO: 6 or 8 and stringent. DNA that can hybridize under various conditions is included. The DNA capable of hybridizing in this manner may be any DNA as long as the protein encoded by the DNA has the ability to inhibit the DNA gyrase activity. Such DNA is SEQ ID NO: 6.
Or, it has a homology of 70% or more, preferably 80% or more, and more preferably 90% or more with the nucleotide sequence shown by 8. As such DNA,
It includes mutant genes found in nature, artificially modified mutant genes, and homologous genes derived from heterologous organisms.

In the present invention, hybridization under stringent conditions is usually carried out at 37 to 42 ° C. in 5 × SSPE (5 times concentration SSPE) or a hybridization solution having a salt concentration equivalent thereto. Performed under temperature conditions for about 12 to 18 hours, pre-washed with 5xSSPE or a solution having a salt concentration equivalent thereto, etc., if necessary, and thereafter, in a solution having a salt concentration equivalent to 1xSSPE or 50 to 65 ° C. It can be carried out by performing washing under the temperature condition of. Further, in order to obtain higher stringency, washing may be performed in a solution having a lower salt concentration, for example, 0.1 × SSPE or a solution having a salt concentration equivalent thereto.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention.

In the following examples, each operation is "Molecular Clonin" unless otherwise specified.
g "(Sambrook, J., Fritsch, EF and Maniatis, T .;
(Published by Cold Spring Harbor Laboratory Press in 1989), or when using commercially available reagents and kits, they were used according to the instructions for the commercially available products.

[0036]

【Example】

Example 1 E. coli DNA gyrase inhibitory protein (DG
I) Isolation and identification (1) Escherichia coli KL1 as a culture strain of the bacterium
The culture was performed as follows using 6 strains. (NH ₄ ) ₂
SO ₄ 1g, KH ₂ PO ₄ 3g, K ₂ HPO ₄ 5.25
g, sodium citrate dihydrate 0.57 g, MgS
Dissolve 0.12 g of O ₄ in 1000 ml of pure water and sterilize it. Separately sterilize casein hydrolyzate and glucose to 1%.
Was added to prepare a medium. This medium (15
0 ml) into a 500 ml Erlenmeyer flask and 10 ml
The culture solution (of overnight culture) was added, and the mixture was vortexed (160 rpm) at 37 ° C. Late log phase (OD60
0 about 1.0) The culture solution was rapidly cooled and then centrifuged at 4 ° C (5000 r
pm x 10 min) to collect the bacteria. Add this to TED buffer (10 mM Tris-HCl (pH 7.5), 1
2 with mM EDTA, 1 mM dithiothreitol)
After washing twice, weighing the wet weight, it was suspended in an equal volume of TED buffer, dispensed in approximately 20 ml aliquots, and stored at -80 ° C.

(2) Purification of Escherichia coli DNA gyrase Purification of DNA gyrase was carried out by the method of Aoyama et al. (Antimicrobial Agents and)
Chemotherapy, Volume 32, 104-10.
Page 9, 1989).

That is, the cells stored at −80 ° C. were thawed at room temperature, and 0.5 ml of dithiothreitol 0.5M, 0.5M EDTA 2 ml, 20 ml per 20 ml.
4 ml of 5M KCl and 2 ml of a 30 mg / ml lysozyme solution were sequentially added, and the mixture was slowly shaken at room temperature for 15 minutes. Then, Bridge 58 (manufactured by Sigma) was added to 0.5% and the mixture was stirred in ice for 30 minutes. Ultracentrifugation (35,000 rpm x 1h) at 4 ℃
r) was performed and the supernatant was collected. 20% streptomycin was added to this supernatant to a final concentration of 2% to remove nucleic acids. Then, the mixture was stirred in ice for 30 minutes and centrifuged at 4 ° C. (12,000 rpm × 15 min) to obtain a supernatant. 0.39 g of ammonium sulfate was added to 1 ml of the supernatant, and the mixture was centrifuged (12,000 rpm x
A precipitate was obtained in 20 min). Precipitate 20 ml TE
After suspending in D buffer, it was dialyzed against 4000 ml of TED buffer at 4 ° C. to obtain a crude extract (50 ml). The crude extract is
Novobiocin pre-equilibrated with TED buffer
A Sepharose column (bed volume 30 ml) was loaded. The Novobiocin-Sepharose column is a Sta
edenbauer and Orr's method (Nucleic
Acids Research, Volume 9, 3589-
3603, 1989), epoxy-activated Sepharose 6B (Pharmacia) coupled with novobiocin (Sigma) was used.
After washing the column with 6 volumes of TED buffer, 0.2M
KCl, 2M KCl, 5M urea, 2M KCl-5
M urea, 2M KCl-5M urea (pH 4.0)
Were sequentially eluted with a TED buffer containing each of the above, and fractionated. The eluate fractions (3 ml each) were dialyzed against TED buffer-50% glycerin at 4 ° C. overnight, and the supercoiling activity of each fraction was measured by the method described in Reference Example below.

Fractions in which activity was recognized were fractionated as containing DNA gyrase holoenzyme (complex of subunits A and B). A part of the fraction other than the active fraction was collected, and the diluted active fraction was added to this to test whether or not the supercoiling activity was recovered. The fraction in which the supercoiling activity was recovered was collected as a crudely purified subunit A fraction. Next, this subunit A
A part of the fraction was added to the other fractions to test whether the supercoiling activity was restored. The fraction in which the supercoiling activity was recovered was collected as a crudely purified subunit B fraction. The elution pattern of proteins from the novobiocin-Sepharose column is shown in FIG. The holoenzyme of DNA gyrase (complex of subunits A and B), subunit A, and subunit B are fractions eluted with 5M urea, fractions eluted with 2M KCl, and 2M, respectively.
Obtained in the fraction eluted with KCl + 5M urea.

(3) Purification of subunits A and B of E. coli DNA gyrase Purification of subunits A and B was carried out as follows. Eighty-five ml of the crudely purified subunit A fraction (diluted 5 times with TED buffer) obtained in (2) above was
The column was loaded on a heparin-Sepharose CL-6B (Pharmacia) column (bed volume 15 ml) equilibrated with a TED buffer containing 0% glycerin. After washing this with the same buffer, 0.05M KCl, 0.2M KC
1, 2M KCl, 2M KCl-5M urea, 2M
Fractionation was performed by eluting with 10% glycerin-containing TED buffer containing KCl-5M urea (pH 4.0). The holoenzyme fraction (about 2.5 μl) obtained in (2) above was added to each fraction (about 5 μl), and the supercoiling activity was measured to determine the fraction containing the purified subunit A.

35 ml of the subunit B fraction (diluted 5-fold with TED buffer) obtained in the above (2) was added to 1
Novobiocin-Sepharose column (bed volume 10 m) equilibrated with TED buffer containing 0% glycerin
l) was loaded. After washing with the same buffer, 2M KCl,
Fractionation was performed by eluting with 10% glycerin-containing TED buffer containing 2.5M urea, 5M urea, 2M KCl-5M urea, and 2M KCl-5M urea (pH 4.0). The purified subunit A fraction was added to each fraction, the supercoiling activity was measured, and the purified subunit B fraction was determined. Each purified subunit fraction was dialyzed against TED buffer containing 50% glycerin and stored at -20 ° C.

(4) Isolation of Escherichia coli DNA Gyrase Inhibitory Protein All the fractions obtained by the novobiocin-sepharose column chromatography in (2) above were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for activity. The electrophoretic patterns of the fractions exhibiting the activity and the fractions exhibiting no activity were compared. As a result, No. The fractions 32 and 33 did not show supercoiling activity despite showing the electrophoretic pattern of the protein corresponding to the holoenzyme, and a protein band of 18 kDa was commonly observed in these fractions. (Figure 2).

Fraction No. 32 (0.4 ml) 10%
Sephadex G-75 (column volume 3.5 ml) equilibrated with TED buffer containing glycerol was loaded and gel filtration was performed. After elution with TED buffer and fractionation,
While confirming a protein band of 18 kDa by SDS-polyacrylamide electrophoresis by taking a part of each fraction,
A fraction in which the 8 kDa protein, that is, the DNA gyrase inhibitory protein (DGI) became a single band was collected as purified DGI. As a result, approximately 1.5 μg of purified DGI was obtained from 26.5 g of wet bacterial cells.

(5) DNA Gyrase Inhibitory Activity of DGI A system for measuring supercoiling activity of DNA gyrase described in Reference Example below, that is, relaxed pBR322 DNA
(0.1 μg), subunit A (1 U) and subunit B (1 U) in the reaction solution (15 μl). Three
When 2 fractions (5 μl) were added and reacted, the relaxed plasmid DNA was not converted to the supercoiling type (FIG. 3). From this, DN for 32 fractions
It was confirmed that a substance that inhibits the supercoiling activity of A gyrase was contained. Also, (4) above
It was confirmed that the purified DGI obtained in 1. completely inhibited the supercoiling activity of DNA gyrase at a reaction solution concentration of 8 μg / ml.

(6) Determination of N-terminal amino acid sequence of Escherichia coli-derived DGI The N-terminal amino acid sequence of the purified DGI obtained in (4) above was determined using a peptide sequencer LF3400 (manufactured by Beckman). N-terminal side 1 obtained as a result
The amino acid sequence of 6 residues is shown in SEQ ID NO: 1 in the sequence listing below.

Example 2 Cloning of Escherichia coli dgi gene and determination of nucleotide sequence Nucleic acid sequence data bank EMBL / Genbank / DD
Using BJ, homology search was performed on the N-terminal amino acid sequence of Escherichia coli DGI determined in item (6) of Example 1, and as a result, the N-terminal sequence of DGI was Escherichia coli K-1.
2 strain chromosomal DNA sbcB region (EMBL accession number U
(00009) and the N-terminal amino acid sequence of the expected product YeB. In addition, Ye calculated from the array
The molecular weight of eB was 18,081 Dalton, which was in agreement with the molecular weight of DGI. From these facts, it was considered that the assumed YeB is DGI and the gene encoding DGI (dgi gene) is in the portion containing the translation region of the eyeB gene.

Therefore, E. E. coli KL16 strain chromosomal DNA, the sbcB region (EMBL accession number U0
The region corresponding to the 189th to 1370th bases on the 5'side of 0009) is designated as polymerase chain reaction (PCR).
It was cloned as follows by se chain reaction). An oligonucleotide primer (a sequence of a sense primer is shown in SEQ ID NO: 2 in the sequence listing and a sequence of an antisense primer is shown in SEQ ID NO: 3) in which BamHI recognition sites are incorporated at both ends of the target sequence is designed. It was synthesized with a DNA synthesizer. PCR is 0.5 mg
/ Ml E.I. coli KL16 chromosomal DNA, 800
μM dNTPmixture, 0.66 μM sense primer, 1 μM antisense primer, 2 mM
PCR buffer containing MgCl ₂ , 0.025 U / μl Taq polymerase (composition: 50 mM KCl,
10 mM Tris-HCl (pH 9.0), 1% Tr
The reaction was carried out in itonX-100 (2 at 94 ° C).
Min, 51 ℃ 2 minutes, 72 ℃ 3 minutes 30 cycles), DNA
Was amplified. 1.2 kbp DNA obtained by PCR
The fragment was cleaved with an appropriate restriction enzyme and ligated to the vector plasmid pUC19 (Takara Shuzo), and the resulting recombinant plasmid was E. coli. E. coli JM109 strain. Using these recombinant plasmids, the fluorescent DNA sequencer (manufactured by Hitachi) was used for the dideoxy method to obtain 1.2 kbp DN.
The base sequence of the A fragment was determined. The obtained nucleotide sequence and the amino acid sequence of the protein encoded by the open reading frame in this sequence are shown in SEQ ID NO: 6. This sequence has been registered in the data base of DNA base sequence E. It matched the sequence (yeeB gene) in the sbcB region of E. coli K-12 (EMBL accession number U00009). In addition, the N-terminal amino acid sequence of the protein encoded by the open reading frame matches the N-terminal sequence of DGI, and the DNA and amino acid sequences shown in SEQ ID NO: 6 are E. coli. DN of E. coli dgi gene
It was considered to be the A sequence and the amino acid sequence of DGI.

Example 3 The dgi gene DGI of various bacterial chromosomes was important for bacterial growth and was expected to exist universally. Therefore, the dg obtained in Example 2 above
The DNA fragment of the i gene was used for Southern hybridization with chromosomal DNA of various bacteria.

Southern hybridization is performed by Mol
electrical Cloning (Volume 2, 6.2-
6.19, 9.31-9.46, 1989). Chromosomes derived from various bacteria were prepared by a conventional method and then digested with EcoRI. As the probe, E. coli cloned in (6) of Example 2 above was used. The NdeI digested fragment in the gene derived from E. coli was used after being labeled with ³² P. Hybridization was performed under normal stringent conditions. That is, SSPE (180 mM NaCl, 1 mM ED
Prehybridization solution (5 × SSPE, containing TA, 10 mM sodium phosphate (pH 7.7))
5x Denhardt's solution, 0.5% SDS, 50% formamide) at 42 ° C for 4 hours, after which a labeled probe was added and hybridization was performed at 42 ° C for 14 hours.
The washing was performed under the following three conditions.

Washing conditions 1: 0.1% SDS, 0.1% SSPE 65 ° C. Washing conditions 2: 0.1% SDS, 0.1% SSPE 52 ° C. Washing conditions 3: 0.1% SDS, 1 × SSPE 52 ° C. The strength of hybridization was determined as follows.

+++: Autoradiography after washing under washing condition 1 and clear hybridizing band ++; Autoradiography after washing under washing condition 2 and clear hybridizing band +; washing After washing under condition 3, autoradiography was carried out, and the hybridization band was clear. The results are shown in Table 1 below.

[0052]

[Table 1]

As shown in Table 1, genus Shigera, C
genus itrobacterium, genus Pseudomonas,
Bacillus, Enterococcus, S
In the bacterium of the genus taphylococcus, Esche
dgi gene fragment derived from richia coli (NdeI corresponding to the 255th to 815th positions in SEQ ID NO: 6)
The fragment was found to hybridize with the fragment under stringent conditions.

That is, it is considered that homologous dgi genes are present in these microorganisms, and in particular Shige
Genes derived from the genera ra and Citrobacter are E
It was considered to have extremely high homology to those derived from the genus Scherichia. Since the gene homologous to the dgi gene is distributed in many bacterial species in this way,
It is suggested that DGI is important for bacterial growth.

Example 4 Escherichia coli DG by gene recombination
Large-scale expression and purification of I (1) Preparation of vector for DGI expression In order to prepare a large amount of DGI protein, a vector expression plasmid pLEX (manufactured by Invitrogen) was used to construct a DGI expression system as follows. . The expression vector pLEX system controls the expression of a target gene by controlling transcription using tryptophan as an inducer. That is, in the absence of tryptophan, the cl repressor protein is expressed from the λl gene under the trp promoter, and the cl repressor protein binds to the operator region of the PL promoter on the vector and is linked to the downstream region of the PL promoter. Suppress the transcription of. On the other hand, in the presence of tryptophan, a tryptophan-repressor complex is formed, and this complex strongly binds to the trp operator and blocks the expression of the cl repressor protein, and the cl repressor dissociates from the PL promoter, resulting in the gene of interest. Transcription occurs.

As a fragment to be incorporated into the plasmid pLEX, restriction enzyme NdeI recognition sites were added to both ends of the dgi gene region (the fragment corresponding to the 255th to 815th bases of SEQ ID NO: 6) whose nucleotide sequence was determined in Example 2 above. The fragment thus prepared was prepared by PCR. PCR template DN
As A, E. E. coli KL16 strain chromosomal DNA was used to synthesize the primers (the sequence of the sense primer is shown in SEQ ID NO: 4 and the sequence of the antisense primer is shown in SEQ ID NO: 5) with a DNA synthesizer. PCR obtained
The product was ligated to vector plasmid pT7BlueT (Novagen), and E. E. coli JM109 was introduced to prepare a recombinant plasmid. The resulting recombinant plasmid was cleaved with NdeI, separated by agarose gel electrophoresis, and a 473 bp NdeI fragment was purified from the gel, which was ligated to pLEX. This was electroporated by the E. coli GI724 (Invitro
gen, USA) to prepare a recombinant plasmid, and the insertion direction of the insert fragment was determined by the EcoRV cleavage pattern, and the insert fragment containing the dgi gene was inserted in the correct direction (sense direction). Replacement plasmid p
Obtained CA17. For electroporation, an electrocell manipulator 600 (manufactured by BM Equipment Co., Ltd.) was used, potential difference 2.25 kV, electric resistance 129.
It was performed under the condition of Ω. GI724 transformed with the recombinant plasmid pCA17 in which the insert fragment containing the dgi gene was inserted into the vector plasmid pLEX in the forward direction.
Strain, namely E. coli GI724 (pCA17)
The strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology under the deposit number FERM BP-16133.

(2) Large-scale expression of Escherichia coli DGI E. coli obtained in the above item (1). coli GI724 (pC
A17) strain, R containing ampicillin 100 μg / ml
M medium (42 mM Na ₂ HPO ₄ , 22 mM KH ₂ P
O ₄ , 8.5 mM NaCl, 18 mM NH ₄ Cl (p
H7.4), 2% casamino acid, 1% glycerol, 1 m
It was shake-cultured in M MgCl ₂ ). When the OD600 value reached about 0.5, tryptophan was added to a final concentration of 100 μg / ml to induce the expression of DGI protein. Then, shaking culture was continued at 37 ° C. for 5 hours. 1 ml of the culture solution was collected and centrifuged to collect the cells. The cells obtained were mixed with 100 μl of SDS buffer (50 mM Tr
is-HCl (pH 6.8), 138 mM SDS,
It was dissolved in 1.5 M glycerin, 280 mM 2-mercaptoethanol), heated at 95 ° C. for 5 minutes to lyse, and stored in ice. For 20 μl of the supernatant, SD
S-polyacrylamide gel electrophoresis (SDS-PAG
Protein expression was examined according to E). The result (Fig. 4), p
Approximately 18 corresponding to DGI was found in the transformant strain with CA17.
Large expression of a kilodalton protein was observed. on the other hand,
Large-scale expression was not observed in the strain transformed with the recombinant plasmid pCA18 or the vector pLEX in which the insert fragment containing the dgi gene was inserted in the reverse direction (antisense direction). In addition, microscopic observation of a strain that expresses a large amount of DGI revealed that it was elongated and elongated, and that cell division was abnormal. From this, DG
Agents that enhance the activity and / or expression of I are considered useful as antibacterial agents.

(3) Purification of E. coli DGI E. E. coli GI724 (pCA17) strain was cultured and expression-induced by tryptophan in the same manner as in (2) above. The bacterial cells obtained by centrifugation were mixed with A solution (30 mM Tris-HCl (pH 7.5), 30 m
(M NaCl) and sonicated in ice. After crushing, centrifugation (4 ° C, 8000 rpm, 20
The resulting supernatant was subjected to ammonium sulfate precipitation at 60% saturation and centrifuged at 17,000 × G for 20 minutes. The resulting pellet was added to 30 ml of TED solution (10 mM Tr
is-HCl pH (7.5), 1 mM EDTA, 1 m
MD TTT) and dialyzed against the same solution. After dialysis, the sample was loaded on a Q-Sepharose fast flow column (0.55 cm ² x 9 cm) (Pharmacia) equilibrated with a TED solution in advance, and a linear concentration gradient of NaCl (from 0.025 M to 0.8 M) was applied. Then, the protein was eluted and fractionated with 140 ml of TED solution (linear velocity 15
0 cm / hr). SDS-PAGE was performed for each fraction, and the fraction in which a protein band of 18 kDa was confirmed was collected. About this fraction, T equilibrated with TED solution
SKgel Toyopearl HW-55 (2.25 cm ² x
32 cm) (manufactured by Tosoh Corporation) was fractionated by gel filtration, and the fraction in which a protein band of 18 kDa was confirmed was 20
It was concentrated by centrifugation to 0 μl. After the concentrated solution was subjected to SDS-PAGE,
Extract the desired DGI protein from the gel and re-select TSKge
Purified DGI after gel filtration with Toyopearl HW-55
(600 μg) was obtained. The amount of protein was calculated by measuring the absorbance at 595 nm using a Coomassie Brilliant Bull-dye solution for protein determination.

Example 5 Measurement of DGI activity The purified DGI obtained in Example 4 was used to measure the DGI activity, that is, the inhibitory activity against the DNA gyrase activity (supercoiling activity). Activity measurement reaction solution (20 mM Tris-HCl (pH 7.5), 2
0 mM KCl, 4 mM MgCl ₂ , 4 mM spermidine, 1.5 mM ATP, 1 mM DTT, 30 μ
g / ml t-RNA, 15 μg / ml BSA, 0.
DNA gyrase subunit A (1U), subunit B (1
U) Add 5 μl of each and add DGI (protein concentration 25,
12.5, 6.25, 3.13, 1.56, 0.78 or 0.39 μg / ml) was added and reacted at 37 ° C. for 1 hour. After adding 3 μl of proteinase K (1 mg / ml) and further incubating at 37 ° C. for 30 minutes to stop the reaction,
After the reaction solution was electrophoresed on 0.8% agarose gel,
Stain with 0.5 μg / ml ethidium bromide solution,
Photographed under UV irradiation. The amount of supercoiled DNA was measured by a densitometer (Shimadzu-wavelength flying spot scanner CS-9000, manufactured by Shimadzu Corporation). DGI
The presence or absence of DNA gyrase inhibitory activity was assayed by comparing the amount of supercoiled DNA when protein was added with the amount of supercoiled DNA when no protein was added. The results are shown in Fig. 5. The supercoiling activity of DNA gyrase was completely inhibited when DGI was added at a concentration of 6 μg / ml or more. If the concentration is lower than this, super-coil type DNA conversion is terminated in an incomplete state, and relaxed type D
A band with a thin tail was detected from NA.

As described above, a DGI activity measuring system could be constructed. In this measurement system, drug solution (or suspension)
By carrying out the reaction with and without addition, and comparing the DGI activity, the action of modulating the DGI activity of the drug (activity enhancing action or activity inhibiting action) can be detected,
Thus, agents that modulate DGI activity can be screened for or identified.

Example 6 Measurement of promoter activity of Escherichia coli dgi gene (1) Preparation of promoter activity measuring plasmid E. In the 5'upstream region of the dgi gene of E. coli, sbc
There is a region (yeC gene) encoding the hypothetical product YeeC in the B region (EMBL accession number U00009). Although both genes are separated by about 122 bp, a palindromic structure that is considered to be a ρ-factor-dependent terminator of yeeC and a -10 and -35 region that is presumed to be the promoter of the dgi gene are typical between the two genes. Various sequences are recognized. Therefore, as described below, a plasmid was prepared in which a DNA fragment containing the promoter region of the dgi gene was ligated to the lac gene, and a system capable of measuring the dgi promoter activity using β-galactosidase activity as an index was constructed.

A 1.2 kbp DN containing the promoter region of the dgi gene, the transcription initiation region and the 5'side of the translation region.
DN in which BamHI recognition sites are added to both ends of the A fragment (fragment corresponding to the 3rd to 1184th bases of SEQ ID NO: 6)
The A fragment was prepared by PCR. As the primers, two kinds of oligonucleotides each having a BamHI recognition sequence added to the sequences at both ends of the target region were synthesized and used.
Chromosomal DNA of Escherichia coli KL16 strain was used as a template. The fragment obtained by PCR was digested with BamHI and RsaI.
Double cleavage at 283 bp D consisting of 33 bp on the 5'side of the putative promoter region, transcription initiation region and translation region.
The NA fragment was prepared. Vector plasmid pLGlac
Z7 (Plasmid Vol. 32, 233-237
(Page, 1994) with BamHI and blunt-ended with the previously obtained 283 bp DNA fragment.
Both were ligated to produce a recombinant plasmid. Thus, a recombinant plasmid in which a fragment containing the dgi promoter was ligated in the same direction upstream of the lacZ gene, that is, a plasmid pCA15 expressing a fusion protein of the N-terminal 11 amino acid residues of the DGI protein and β-galactosidase was obtained. . In addition, a recombinant plasmid pCA16 in which a fragment containing the dgi promoter was inserted in the reverse direction was also obtained.

(2) Measurement of dgi promoter activity E. The recombinant plasmid pCA15 obtained in (1) above was transformed into E. coli JM109 strain with E. coli. coli JM10
Using the transformant introduced in No. 9, it was measured as follows. An overnight culture of the strain was inoculated into 10 ml of LB medium containing 50 μg / ml kanamycin so that the OD600 value was 0.1, and shake culture was carried out at 37 ° C. The turbidity at 600 nm was measured every hour, and about 0.3 to 0.5 ml of the culture solution was collected. The collected culture solution was diluted to an OD600 value of 0.1, and 1 ml was collected and centrifuged (14000 rpm, 1 minute).
Then, the obtained bacterial cells were mixed with 1 ml of Z buffer (60 mM Na
₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 10 mM KC
1, 1 mM MgSO ₄ , 50 mM β-mercaptoethanol). After ultrasonically crushing this, centrifuging and collecting 600 μl of the supernatant liquid as a sample solution,
The β-galactosidase activity of each sample solution was measured as follows. 600 μl of sample solution for 5 minutes at 28 ° C
After incubating at 0 ° C., a solution of reaction substrate ONPG (O-nitrophenyl-β-D-galactopyranoside) (4 m
(g / ml) (200 μl) was added, the mixture was kept warm for 5 minutes to cause reaction, and then 500 μl of 1M sodium carbonate was added to stop the reaction. After completion of the reaction, the absorbance at 420 nm and 550 nm was measured, and the method of Sambrook et al.
color Cloning: A Laborator
y Manual, Second Edition, Cold Spring
Harbor Laboratory Press, P
The β-galactosidase activity and the specific activity per 1 μg of the protein amount were calculated according to Lainview, New York, 1989). The amount of protein in the sample solution was measured using a CBB dye solution for protein determination.

Dgi promoter activity (β-
The result of measuring the temporal change of galactosidase (using the expression of galactosidase as an index) is shown in FIG. The β-galactosidase activity per 1 μg of cell protein amount showed the lowest value in the logarithmic growth phase (OD600: 0.5) after 2 hours of culture, and then reached the stationary phase (OD600: 1.2) for 6 hours. It turned out to rise to later.

Plasmids pCA15, pCA16 and p
E. coli in which each LGlacZ7 was introduced. coli JM10
For 9 strains, the dgi promoter activity was measured in the same manner as above. As a result, the β-galactosidase activity per 1 μg of bacterial cell protein after 18 hours of culture was 21
6, 14 and 0 units / μg. Thus, in E. coli harboring pCA15, a stronger β compared to control plasmids (pCA16 and pLGlacZ7).
-The expression of galactosidase was observed, which indicates that
It was confirmed that by using the plasmid pCA15, the dgi promoter activity can be easily measured using the β-galactosidase activity as an index.

As described above, a dgi promoter activity measuring system could be constructed. In this measurement system, culture was performed with and without addition of a drug solution (or suspension), and
By comparing the promoter activities, it is possible to detect the action of the drug that modulates dgi gene expression (expression enhancing action or expression inhibitory action), and thus it is possible to screen or identify a drug that modulates DGI expression. it can.

Example 7 Control of Escherichia coli dgi Gene Expression by Antisense RNA (1) Expression Control of dgi Gene by Introduction of Antisense RNA Expression Plasmid A fragment containing the translation region of the dgi gene (the 255th to 728th positions in SEQ ID NO: 6) (A fragment corresponding to the base of 1) was inserted downstream of the tryptophan (trp) promoter of the vector plasmid pLEX (manufactured by Invitrogen) to prepare an antisense expression vector.

E. E. coli GI724 strain. The obtained antisense RNA-expressing strain was cultured under conditions with or without the addition of tryptophan, and the successive changes in viable cell count and turbidity (OD600) were measured. The results are shown in Fig. 7. When expression induction by addition of tryptophan was not carried out, the number of bacteria after 1 hour was 1 × 10 ⁹
cfu / ml, turbidity increased to 1.0. On the other hand, tryptophan was added to the dgi gene antisense RNA.
When the expression of is induced, the viable cell count is 8 hours after addition,
Although a slight increase tendency was seen from 4 × 10 ⁷ cfu / ml to 1 × 10 ⁸ cfu / ml, it was 5 × 1 after 24 hours.
0 was reduced to ⁶ cfu / ml. Also, the OD600 value is
The value increased to 0.4 8 hours after the addition, but remained a constant value of 0.5 even after 24 hours. Also, antisense R
Elongation of the bacteria was observed in the NA-expressing strain. It has been reported that such morphological abnormalities are also observed when Escherichia coli is treated with a new quinolone type gyrase inhibitor (Takeshi Nishino et al., Chemotherapy, Volume 41 (s-5), 50-66). P., 1993). From the above results, it is considered that in E. coli in which dgi antisense RNA expression was induced, the dgi gene expression was suppressed and the normal growth of the bacterium was inhibited. That is, a drug that suppresses dgi gene expression is considered to be useful as an antibacterial agent.

Example 8 Polyclonal antibody against Escherichia coli DGI protein (1) Preparation of anti-DGI antiserum Solution 2 of purified DGI obtained in the above item (4) of Example 3
25 μg of Gerb adjuvant (manufactured by Nacalai Tesque, Inc.) was added to 00 μl (corresponding to 100 μg), and the mixture was shaken for 5 minutes at room temperature to mix thoroughly, and then subcutaneously administered to the back of a rabbit to perform the first immunization. One week and three weeks after the initial immunization, the same administration was performed to perform booster immunization. After immunization, a small amount of blood was collected from the ear vein to confirm the antibody titer. When an increase in serum antibody titer was confirmed, about 50 ml of blood was collected from the ear artery. The obtained blood is 37 ° C for 1 hour,
Subsequently, the mixture was allowed to stand at 4 ° C. overnight and then centrifuged to obtain serum. The obtained antiserum was filtered through a filter (pore size 0.45 μm)
Then, the mixture was dispensed and stored at -80 ° C.

(2) Purification of anti-DGI antibody (IgG) and dot blot assay EZ-SEP (trade name, Pharmacia, antibody purification kit) was used to purify the IgG fraction from the antiserum. It was The IgG concentration of the purified antibody was measured by the dilution dot blot method (anti-peptide antibody experiment protocol, Shujunsha, Shinobu Oumi, et al., Pp. 73-74) and was found to be 300 mg / ml.

In addition, the minimum amount of antigen protein that can be recognized by using this antibody was assayed by dot blot assay. That is, 20, 10, 5,
2.5 and 1.25 μg / ml of purified DGI at a concentration of 0.
After spotting 5 μl and drying it, 20 mg / m
1BSA-containing TBS (0.15M NaCl, 20 mM
Blocked in Tris-HCl (pH 7.5)). This membrane was immersed in an anti-DGI antibody (1000-fold diluted) diluted with BSA-containing TBS, and incubated at 37 ° C for 1 hour. After washing this membrane, anti-rabbit IgG antibody (Gi labeled with horseradish peroxidase (HRP)
bco. After incubating with BRL) at 37 ° C for 0.5 hour, antibody binding was detected by peroxidase staining using diaminobenzidine (DAB) as a substrate. As a result, the minimum antigen (DGI) protein amount that this antibody could recognize was about 5 ng.

(3) Detection of Escherichia coli DGI by Western Blotting Escherichia coli highly expressing DGI (E. coli GI724 (pCA17) strain obtained in Example 4) was ultrasonically disrupted and then obtained by centrifugation. The supernatant was separated by SDS-polyacrylamide gel electrophoresis. The gel after migration was transferred to a PVDF (polyvinylidene difluoride) film (manufactured by Millipore). The transferred PVDF membrane was stained with Coomassie Brilliant Blue to confirm the protein migration pattern, and then decolorized with methanol to give 20 mg / ml.
Blocked in TBS containing BSA. BS this film
After immersing in an anti-DGI antibody diluted with A-containing TBS (1000-fold dilution) to react, peroxidase staining was carried out using HRP-labeled anti-rabbit IgG antibody and substrate DAB as in the dot blot assay of (2) above. It was As a result, as shown in FIG. 8, a protein having a molecular weight of 18 kDa corresponding to DGI could be specifically detected. From this, it was considered possible to detect and quantify DGI using the obtained anti-DGI antibody (IgG).

Reference Example A method for measuring supercoiling activity of DNA gyrase Antimicrobial Agents and
Chemotherapy Volume 32, 104-10
It was carried out as follows according to the method described on page 9, 1988.

0.5 μg of pBR322 DNA (super helix type, manufactured by Takara Shuzo) and 1 μl of topoisomerase I (manufactured by Takara Shuzo) (6 U / μl) were added to a reaction solution (35 mM Tr).
is-HCl (pH 8.0), 72 mM KCl, 5 m
M MgCl ₂ , 5 mM DTT, 5 mM spermidine, 0.01% bovine serum albumin) 20 μl,
After reacting at 37 ° C for 2 hours, relaxed pBR322DNA
Was prepared. This relaxed pBR322 DNA 0.1 μ
Reaction solution for supercoiling activity measurement containing 20 g (20 m
M Tris-HCl (pH 7.5), 20 mM KC
1, 4 mM MgCl ₂ , 4 mM spermidine, 1.
5 mM ATP, 1 mM dithiothreitol, 30 μ
g / ml t-RNA (derived from yeast), 15 μg / ml
5 μl of bovine serum albumin), 5 μl of sample solution, DN
A gyrase subunit A (1U / 1.25μl)
5 μl and subunit B (1 U / 1.25 μl) 5
μl was added, and the mixture was reacted at 37 ° C. for 1 hour. To this, add 3 μl of proteinase K (1 mg / ml), and add 37
The reaction was stopped by keeping the temperature at 30 ° C. for 30 minutes. After the reaction,
0.8% agarose gel electrophoresis (running buffer TAE;
40 mM Tris-acetate, 1 mM EDT
After electrophoresis in A), the gel was stained with 0.5 μg / ml ethidium bromide solution and then photographed under UV irradiation. The presence or absence of DNA gyrase inhibition was assayed by comparing the amount of supercoiled DNA when the sample was added and when the sample was not added. Regarding the enzyme activity, the amount of enzyme required to convert 50% of the amount of relaxed plasmid DNA into supercoiled DNA was defined as 1 unit (U).

Example 9 Cloning of dgi gene of Shigella microorganism As described in Example 3 above, Shigella (Shigell)
a genus a) has E. An EcoRI fragment that strongly hybridizes with the dgi gene derived from E. coli was observed, and the size of the fragment was found to be about 7 kb. So Shigella dys
enteriae) The chromosome of IID633 is EcoRI
It was cleaved and subjected to agarose electrophoresis to recover a DNA fragment of about 7 kb. The obtained DNA fragment was Ec of pUC19
The resulting recombinant plasmid ligated to the oRI cleavage site was transformed into E. E. coli JM109 strain. E. coli derived d
Colony hybridization was performed using a DNA fragment containing the gi gene as a probe, and positive colonies 1
As a result of analyzing 0 strain, 6 strains were found to have 7.5 kb on pUC19.
Carried the EcoRI DNA fragment. The inserted fragment was cleaved with various restriction enzymes and analyzed by hybridization with a DNA fragment containing the E. coli dgi gene. As a result, it was considered that the Shigella genus dgi gene was present on the 1.5 kb AvaI fragment.

To determine the nucleotide sequence, various deletion plasmids were prepared from the plasmid containing this DNA fragment, and using these plasmids, the nucleotide sequence was determined by the dideoxy method using a fluorescent DNA sequencer (HQ, SQ5500). It was determined. The 1444 bp sequence was determined.
Comparing the 1444 bp DNA base sequence of Shigella dicenterier with the sequence around the dgi gene of Escherichia coli, high homology of 98.3% was observed over the entire region, and the region from the 699th base to the 1169th base was found. It was found that the open reading frame (ORF) corresponding to is the ORF of the dgi gene. A sequence considered to be a promoter and a ribosome binding region was found upstream of the ORF, and a sequence presumed to be a row-independent terminator was found following the ORF.
14 including the dgi gene derived from Shigella dicenterier
The DNA sequence of the 44 bp fragment and the amino acid sequence of DGI encoded therein are shown in SEQ ID NO: 8. Also,
The amino acid sequence is shown in SEQ ID NO: 9. Shigella dicenteriae DGI is a polypeptide consisting of 157 amino acid residues like E. coli DGI, and its molecular weight was estimated to be about 18 kilodaltons. The amino acid sequences of both DGIs are 9
It showed a high homology of 6.8%.

[0077]

INDUSTRIAL APPLICABILITY The DNA gyrase inhibitory protein (DGI) of the present invention can inhibit normal growth of bacteria by modulating its activity or its expression. By using the DNA gyrase inhibitory protein (DGI) of the present invention and its gene (dgi gene),
The activity or the dgi gene promoter activity can be measured, and these measurement methods are useful as screening methods and identification methods for agents that modulate DGI activity or dgi gene expression. These screening methods and identification methods are useful for developing antibacterial agents and pesticides based on new modes of action. Furthermore, antisense RNA or DNA for the dgi gene is useful as a drug that suppresses DGI expression, and anti-DGI antibody is used for detecting DGI.
It can be used for quantification.

[0078]

[Sequence list]

SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: linear Sequence type: Protein Fragment type: N-terminal fragment origin Organism name: Escherichia coli Share name: KL16 Array Met Asn Tyr Glu Ile Lys Gln Glu Glu Lys Arg Thr Val Ala Gly Phe   1 5 10 15 16 .

SEQ ID NO: 2 Sequence length: 30 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acid Synthetic DNA sequence CTGGATCCAT CAGCGGGTAG GGGAAATTGA 30 .

SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GTGGATCCCA GACTAACATC AGCGGTAACG 30 .

SEQ ID NO: 4 Sequence length: 24 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CACATATGAA CTACGAGATT AAGC 24 .

SEQ ID NO: 5 Sequence length: 28 Sequence type: Nucleic acid Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CACATATGAG CGAAAAAATT GAAAGGCG 28 .

SEQ ID NO: 6 Sequence length: 1224 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Escherichia coli
coli) strain name: KL16 sequence CCATCAGCGG GTAGGGGAAA TTGAACTTTA CGACCGTGAT AAACAGGTGG CGCACTGGCC 60 GCTGGTTACC CTGGAATCTG TCGGGGAAGG CAGCATGTTT TCTCGCCTGA GTGATTATTT 120 CCACCATAAG GCCTGACCTT TCTTTTGCAG CAGACTGGCA GGAGTGCGAG TCTGCTCGCA 180 TAATCAACAC TCATTCCTTG TGGTTTTAAT TTTGCAACTA TACTGTATAT AAAAACAGTA 240 TCAATGGAGG CGTC 254 ATG AAC TAC GAG ATT AAG CAG GAA GAG AAA CGT ACC GTT GCA GGT 299 Met Asn Tyr Glu Ile Lys Gln Glu Glu Lys Arg Thr Val Ala Gly 1 5 10 15 TTC CAT CTC GTT GGC CCG TGG GAA CAG ACG GTA AAG AAA GGC TTT 344 Phe His Leu Val Gly Pro Trp Glu Gln Thr Val Lys Lys Gly Phe 20 25 30 GAG CAG TTG ATG ATG TGG GTA GAT AGC AAA AAT ATT GTG CCG AAG 389 Glu Gln Leu Met Met Trp Val Asp Ser Lys Asn Ile Val Pro Lys 35 40 45 GAG TGG GTT GCT GTC TAT TAC GAC AAT CCA GAT GAA ACA CCC GCC 434 Glu Trp Val Ala Val Tyr Tyr Asp Asn Pro Asp Glu Thr Pro Ala 50 55 60 GAA AAA TTA CGC TGC GAC ACC GTC GTG ACG GTG CCG GGT TAC TTT 479 Glu Lys Leu Arg Cys Asp Thr Val Val Thr Val Pro Gly Tyr Phe 65 70 75 ACG CTT CCC GAA AAC AGT GAG GGC GTC ATT CTG ACA GAA ATT ACA 524 Thr Leu Pro Glu Asn Ser Glu Gly Val Ile Leu Thr Glu Ile Thr 80 85 90 GGT GGT CAG TAT GCG GTG GCG GTA GCT CGT GTA GTC GGT GAT GAT 569 Gly Gly Gln Tyr Ala Val Ala Val Ala Arg Val Val Gly Asp Asp 95 100 105 TTT GCT AAA CCC TGG TAT CAG TTC TTT AAT AGT CTC TTG CAG GAC 614 Phe Ala Lys Pro Trp Tyr Gln Phe Phe Asn Ser Leu Leu Gln Asp 110 115 120 AGT GCT TAT GAA ATG TTA CCA AAG CCC TGC TTC GAG GTT TAT TTG 659 Ser Ala Tyr Glu Met Leu Pro Lys Pro Cys Phe Glu Val Tyr Leu 125 130 140 AAC AAT GGC GCG GAA GAT GGG TAC TGG GAT ATC GAA ATG TAT GTT 704 Asn Asn Gly Ala Glu Asp Gly Tyr Trp Asp Ile Glu Met Tyr Val 140 145 150 GCG GTG CAG CCA AAA CAT CAC 725 Ala Val Gln Pro Lys His His 155 157 TAATTCATCT CAGGGCGTAGTTTG CTGCCGGGAA ATTACACTGT TCCCGGTTTG 845 TCCGTCGGAT AATTCAGAGG CGCGCCTTCT GGCCGACAGA TGAGTTATGA GCGCTTTTAA 905 TCTCATTACG GAGTTTCTGC GTGCGTGCCG ATAA GTCATT AAGCCCGTTT GAAATCCGGG 965 TATACCGCCA TTACCGCATT GTGCATGGTA CTCGGGTCGC GCTGGCATTC CTGCTCACTT 1025 TTCTCATTAT CCGCCTGTTT ACTATCCCGG AAAGCACCTG GCCGCTGGTC ACCATGGTGG 1085 TGATTATGGG GCCAATCTCG TTCTGGGGTA ACGTTGTCCC TCGCGCCTTT GAGCGTATTG 1145 GCGGTACGGT GTTGGGTTCG ATTTTAGGTC TTATCGCTCT GCAACTGGAG TTAATCTCGT 1205 TACCGCTGAT GTTAGTCTG 1224.

SEQ ID NO: 7 Sequence length: 157 Sequence type: Amino acid Topology: linear Sequence type: Protein origin Organism name: Escherichia coli Share name: KL16 Array Met Asn Tyr Glu Ile Lys Gln Glu Glu Lys Arg Thr Val Ala Gly   1 5 10 15 Phe His Leu Val Gly Pro Trp Glu Gln Thr Val Lys Lys Gly Phe                  20 25 30 Glu Gln Leu Met Met Trp Val Asp Ser Lys Asn Ile Val Pro Lys                  35 40 45 Glu Trp Val Ala Val Tyr Tyr Asp Asn Pro Asp Glu Thr Pro Ala                  50 55 60 Glu Lys Leu Arg Cys Asp Thr Val Val Thr Val Pro Gly Tyr Phe                  65 70 75 Thr Leu Pro Glu Asn Ser Glu Gly Val Ile Leu Thr Glu Ile Thr                  80 85 90 Gly Gly Gln Tyr Ala Val Ala Val Ala Arg Val Val Gly Asp Asp                  95 100 105 Phe Ala Lys Pro Trp Tyr Gln Phe Phe Asn Ser Leu Leu Gln Asp                 110 115 120 Ser Ala Tyr Glu Met Leu Pro Lys Pro Cys Phe Glu Val Tyr Leu                 125 130 135 Asn Asn Gly Ala Glu Asp Gly Tyr Trp Asp Ile Glu Met Tyr Val                 140 145 150 Ala Val Gln Pro Lys His His                 155 157 .

SEQ ID NO: 8 Sequence length: 1444 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Shigella dysenteri
ae) strain name: IID633 sequence CCGAGTTTTA TCATATGTAC AGTGAGAAAA GTCTCACCTG GAACGGTATC ACCCAGCAAA 60 ACCGTAACGG GTTATTGTGG GATAAAACCA TGAATGTTGA CGGCCTGAAA ACGGGTCATA 120 CTTCTGGTGC CGGGTTTAAT CTCATTGCTT CGGCTGTCGA TGGGCAGCGT CGTCTCATTG 180 CAGTGGTAAT GGGGGCTGAC AGTGCAAAAG GTCGTGAGGA AGAGGCAAGA AAATTACTGC 240 GTTGGGGGCA ACAAAACTTT ACTACGGTGC AAATTTTGCA CCGTGGGAAA AAGGTCGGTA 300 CGGAACGCAT CTGGTATGGC GATAAAGAAA ATATCGACCT GGGAACGGAA CAAGAGTTCT 360 GGATGGTGCT ACCGAAAGCC GAAATTCCAC ATATCAAAGC CAAATATACC CTTGATGGTA 420 AAGAGCTCAC CGCGCCAATT AGCGCCCATC AGCGGGTAGG GGAAATTGAA CTTTACGACC 480 GTGATAAACA GGTGGCGCAC TGGCCGCTGG TTACCCTGGA ATCTGTCGGG GAAGGCAGCA 540 TGTTTTCTCG CCTGAGTGAT TATTTCCACC ATAAGGCCTG ACCTTTCTTT TGCAGCAGAC 600 TGGCAGGAGT GCGAGTCTGC TCGCATAATC AACACTCATT CCTTGTGGTT TTAATATCGC 660 AATTATACTG TATATAAAAA CAGTGTTGAT GGAGCGTC 698 ATG AAC TAC GAG ATT AAG CAG GAA GAT AAA CGT ACC GTT GCA GGT 743 Met Asn Tyr Glu Ile Lys Gln Glu Asp Lys Arg Thr Val Ala Gly 1 5 10 15 TTC CAT CTC GTT GGC CCG TGG GAA CAG ACG GTA AAG AAA GGC TTT 788 Phe His Leu Val Gly Pro Trp Glu Gln Thr Val Lys Lys Gly Phe 20 25 30 GAG CAG TTG ATG ATG TGG GTA GAT AGC AAA AAT ATT GTG CCG AAG 833 Glu Gln Leu Met Met Trp Val Asp Ser Lys Asn Ile Val Pro Lys 35 40 45 GAG TGG GTT GCT GTC TTT TAC GAC AAT CCA GAT GAA ACA CCC GCC 878 Glu Trp Val Ala Val Phe Tyr Asp Asn Pro Asp Glu Thr Pro Ala 50 55 60 GAA AAA TTA CGC TGC GAC ACC GTC GTG ACG GTG CCG AAT AAC TTT 923 Glu Lys Leu Arg Cys Asp Thr Val Val Thr Val Pro Asn Asn Phe 65 70 75 ACG CTC CCC GAA AAC AGT GAG GGC GTC ATT CTG ACA GAA ATT TCA 968 Thr Leu Pro Glu Asn Ser Glu Gly Val Ile Leu Thr Glu Ile Ser 80 85 90 GGT GGT CAG TAT GCG GTT GCG GTG GCT CGT GTA GTC GGT GAT GAT 1013 Gly Gly Gln Tyr Ala Val Ala Val Ala Arg Val Val Gly Asp Asp 95 100 105 TTT GCT AAA CCC TGG TAT CAG TTC TTT AAT AGC CTC TTG CAG GAC 1058 Phe Ala Lys Pro Trp Tyr Gln Phe Phe Asn Ser Leu Leu Gln Asp 110 115 120 AGT GCT TAT GAA ATG TTA CCA AAG CCC TGC TTC GAG GTT TAT TTG 1103 Ser Ala Tyr Glu Met Leu Pro Lys Pro Cys Phe Glu Val Tyr Leu 125 130 135 AAC AAT GGC GCG GAA GAT GGG TAC TGG GAT ATC GAA ATG TAT GTT 1148 Asn Asn Gly Ala Glu Asp Gly Tyr Trp Asp Ile Glu Met Tyr Val 140 145 150 GCG GTG CAG CCA AAA CAT CAC 1169 Ala Val Gln Pro Lys His His 155 157 TAATTCATCT CAGGGCGGTG TGTTAACGCG ATGACCACTC TTTTTTTGAA AGCGAAAAGA 1229 GTAAGATGCG CCTTTCAATT TTTTCGCTCC TGCCGGGAAA TTACACTGTT CCCGGTTTGT 1289 CCGTCGGATA ATTCAGAGGC GGGCCTTCTG GCCGACAGAT GAGTTATGAG CGCTTTTAAT 1349 CTTCATTACG GAGTTTCTGC GTGCGTGCCG ATAAGTCATT AAGCCCGTTT GAAATCCGGG 1409 TATACCGCCA TTACCGCATT GTGCATGGTA CTCGG 1444 .

SEQ ID NO: 9 Sequence Length: 157 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Origin Organ Name: Shigella dysenteri
ae) Strain name: IID633 Sequence Met Asn Tyr Glu Ile Lys Gln Glu Asp Lys Arg Thr Val Ala Gly 1 5 10 15 Phe His Leu Val Gly Pro Trp Glu Gln Thr Val Lys Lys Gly Phe 20 25 30 Glu Gln Leu Met Met Trp Val Asp Ser Lys Asn Ile Val Pro Lys 35 40 45 Glu Trp Val Ala Val Phe Tyr Asp Asn Pro Asp Glu Thr Pro Ala 50 55 60 Glu Lys Leu Arg Cys Asp Thr Val Val Thr Val Pro Asn Asn Phe 65 70 75 Thr Leu Pro Glu Asn Ser Glu Gly Val Ile Leu Thr Glu Ile Ser 80 85 90 Gly Gly Gln Tyr Ala Val Ala Val Ala Arg Val Val Gly Asp Asp Asp 95 100 105 Phe Ala Lys Pro Trp Tyr Gln Phe Phe Asn Ser Leu Leu Gln Asp 110 115 120 Ser Ala Tyr Glu Met Leu Pro Lys Pro Cys Phe Glu Val Tyr Leu 125 130 135 Asn Asn Gly Ala Glu Asp Gly Tyr Trp Asp Ile Glu Met Tyr Val 140 145 150 Ala Val Gln Pro Lys His His 155 157

[Brief description of drawings]

FIG. 1 is a view showing an elution pattern of DNA gyrase by novobiocin-sepharose column chromatography.

FIG. 2 is a photograph of SDS-polyacrylamide gel electrophoresis of fractions obtained by novobiocin-sepharose column chromatography.

FIG. 3 Fraction No. 32 is a photograph of agarose gel electrophoresis showing disappearance of DNA gyrase activity by 32.

FIG. 4 SDS- showing large expression of DGI in E. coli
Photograph of polyacrylamide gel electrophoresis.

FIG. 5 is a photograph of agarose gel electrophoresis showing the inhibitory effect of the purified 18 kD protein on the DNA gyrase supercoiling activity.

FIG. 6 is a view showing changes in dgi promoter activity during the growth stage of Escherichia coli.

FIG. 7 shows the effect of antisense RNA expression of the dgi gene on the growth of Escherichia coli.

FIG. 8 is an electrophoretic photograph showing detection of Escherichia coli DGI by Western blotting using an anti-DGI antibody.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification FI A61K 38/55 A61P 31/04 48/00 C07K 14/245 A61P 31/04 14/25 C07K 14/245 C12N 15/00 ZNAA 14 / 25 A61K 37/64 (56) References Mol. Gen. Genet. , March 20, 1996, Vol. 250, No. 5, p. 593-60 Molecular Microbiology, 1995, Vol. 18, No. 2, p. 301-311 Journal of Molecule Biology, 1984, Vol. 174, No. 4, p. 605-625 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS / WPI (DIALOG) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq PubMed

Claims (9)

(57) [Claims] 1. The following (1), (2), (3),
A protein selected from the group consisting of (4), (5) and (6), which has an action of modulating the DNA gyrase inhibitory activity of a protein having the ability to inhibit the DNA gyrase activity of bacteria. A method for identifying a pharmaceutical compound, which comprises assaying. (1) A protein consisting of the amino acid sequence represented by SEQ ID NO: 7. (2) A protein consisting of the amino acid sequence represented by SEQ ID NO: 7 in which one or several amino acids are deleted, substituted or added. (3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 9. (4) A protein having an amino acid sequence represented by SEQ ID NO: 9 in which one or several amino acids are deleted, substituted or added. (5) A protein encoded by a DNA having the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 8. (6) A protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. 2. The following (1), (2), (3),
Assaying the effect of modulating the expression of a gene encoding a protein which is a protein selected from the group consisting of (4), (5) and (6) and which has the ability to inhibit the DNA gyrase activity of bacteria A method for identifying a pharmaceutical compound, comprising: (1) A protein consisting of the amino acid sequence represented by SEQ ID NO: 7. (2) A protein consisting of the amino acid sequence represented by SEQ ID NO: 7 in which one or several amino acids are deleted, substituted or added. (3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 9. (4) A protein having an amino acid sequence represented by SEQ ID NO: 9 in which one or several amino acids are deleted, substituted or added. (5) A protein encoded by a DNA having the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 8. (6) A protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. 3. The method according to claim 2, which comprises assaying the effect of modulating the expression of a gene by measuring the effect of a pharmaceutical compound on the promoter activity of the gene encoding the protein. 4. The pharmaceutical compound is an antibacterial drug.
Or the method described in 2. 5. The selected protein is (1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7, or (2)
The method according to claim 1 or 2, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 9. 6. The selected protein comprises (1) a DN consisting of the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8.
The protein encoded by A, or (2) a protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. The method described. 7. The gene comprising (1) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 6 or 8 or (2) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 6 or SEQ ID NO: 8. The method according to claim 2, which is a gene consisting of a DNA that hybridizes with the following stringent conditions. 8. The method according to claim 1 or 2, wherein the selected protein is derived from a microorganism belonging to the genus Escherichia. 9. The method according to claim 1 or 2, wherein the selected protein is derived from a microorganism belonging to the genus Shigella.