JPH0678796A - Method for specifying organic compound - Google Patents

Abstract

PURPOSE:To monitor the kind of an organic compound and its existing amount by transducing a luciferase gene below a promoter gene of a decomposition plasmid acting on an organic compound in a specimen, transforming Escherichia coli with the prepared luciferase manifesting type plasmid and dispersing the transformed Escherichia coli into the specimen. CONSTITUTION:A luciferase gene is transduced below a promoter gene of a decomposition plasmid (OCT acting on octane) acting on an organic compound. Escherichia coli is transformed with the prepared luciferase manifesting type plasmid and transformed Escherichia coli is dispersed into a specimen to measure an amount of emitted light. Consequently, the kind of the organic compound and its existing amount can be specified.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for identifying an organic compound.

[0002]

2. Description of the Related Art The action of microorganisms to decompose environmental pollutants, particularly organic compounds centered on aromatic compounds, has been known for a long time. However, since aromatic compounds generally show strong toxicity to microorganisms, there are many difficulties in using microorganisms as they are for the purpose of removing environmental pollutants. Also, many microorganisms can partially decompose complex compounds, but cannot completely decompose them. By the way, most of the genes involved in the decomposition of these substances are present on the chromosome, while the genes responsible for the decomposition of some aromatic compounds and toxic substances are present on the plasmid. Therefore, attempts have been made to breed microorganisms capable of decomposing more complicated compounds and persistent environmental pollutants by combining a degrading plasmid and a degrading gene on the chromosome.

Chakrabarty et al. Have isolated a microorganism capable of degrading 4-chlorobenzoic acid by introducing a TOL plasmid having a complete xylene degrading enzyme system into a 3-chlorobenzoic acid-degrading bacterium (Kellog, ST, Chatterjee, DK). , and Chak
rabarty, AM, Science, 214 , 1133 (1981). This is T
It takes advantage of the extremely broad substrate specificity of the toluic acid oxygenase dominated by the OL plasmid. Therefore, when a gene that controls chemical substance degradation is newly identified, molecular breeding by genetic engineering becomes possible.

By the way, Pseudomonas bacteria are obligately aerobic gram-negative bacilli that grow in soil and sewage.
It has many paths for complete decomposition of aromatic compounds. The feature of this pathway is that it involves reactions by oxygenase such as hydroxylation reaction and ring-opening reaction of benzene ring. Most of the genes involved in the substance degradation of Pseudomonas are present on the chromosome, while some genes involved in the degradation of some aromatic compounds are present on the plasmid. Representative degradation plasmids are shown in Table 1.

[0005]

[Table 1]

As can be seen from Table 1 above, most of the degradation plasmids were isolated from P. putida. Degraded plasmids are generally large and often have self-transmitting ability. Degradative genes form an operon, which exists separately from the region responsible for plasmid replication and transmission. In addition, a regulatory gene that controls the expression of degradation system genes is also present on the degradation plasmid. The degradation plasmid for which the expression control mechanism has been reported in detail is described below.

NAH plasmid NAH7, which is a typical NAH plasmid, is a plasmid having a size of 83 kb. This plasmid has the nahABCDEF peron, which produces salicylic acid from naphthalene, and the two operons of salGHIJKLM, which produces pyruvic acid and acetaldehyde from salicylic acid via a meta-cleavage pathway.
In addition, the positive regulatory region nahR that regulates these operons
Is present between two operons, and it is known that the nahR gene product binds to the promoter region (Pnah, Psal) of each operon and promotes transcription only when salicylic acid is present. There is.

CAN plasmid CAN that produces isobutyric acid from camphor (camphor)
Two operons, camDCAB and camEG, and the regulatory region camR are present on the plasmid (240 kb). This camR gene product has been shown to bind to an operator upstream of camD and suppress operon expression. Negative regulation by camR is characteristic because most Pseudomonas expression genes are positively regulated.

OCT plasmid OCT plasmid (45 which is known to have an enzyme system that oxidizes octane and produces aldehyde through alcohol)
kb) is a non-transmissible plasmid. The existence of the operon alkBAC, which is a degradative gene, and the operon alkRST, which is a regulatory gene, has been shown, and it has been revealed that alkBAC is positively regulated by alkRST.

TOL plasmid The TOL plasmid has an operon xylCAB that produces benzoic acid and m-toluic acid from toluene and m-xylene and an operon xylDLEGF that completely decomposes these into pyruvic acid and acetaldehyde or propionaldehyde. This plasmid, also called pWWO, has a transmissible size of 117 kb. In the regulation of TOL plasmid expression, positive regulatory genes (xylR, x
ylS) has been shown to be involved.

The following has been clarified regarding the control mechanism of the TOL plasmid.

The enzyme group carried by the TOL plasmid is 56k.
localized on a transposon of size b,
Furthermore, these enzyme groups constitute two operons.

As mentioned above, two operons (upper op
eron; xylCAB, metaoperon; xylDLEGF) is positively regulated by two regulatory genes, xylR and xylS, in the presence of inducers such as m-xylene. Where xylR
The product (XylR) activates the expression of each of xylCAB and xylS (Fig. 2 (a)). This activation is performed at the transcriptional stage, and the involvement of NtrA (RpoN) RAN polymerase has been shown. Furthermore, this xylS product (XylS) is xylDLE
A complete degradation system is established by activating the expression of the GF operon (Fig. 2 (b)). The function of NtrA itself was confirmed by genetic analysis of nitrogen-fixing bacteria, but from the beginning it was pointed out that NtrA was involved in Pseudomonas TOL plasmids due to the similarity in the nucleotide sequence of the upstream region involved in gene expression. Recent research shows that the upstream activation region (URS: up
have been shown to be required for complete induction of XylR-mediated genes. XylR
The consensus sequence URS 5'-TGATGATTTGCTNAAATNCACNC-3 'is required for binding to DNA, and there are two repeats upstream of the transcription start of the promoter (Pu) of the upper operon and upstream of the Ps promoter. Furthermore, IH found by chromosomal integration of λ phage
The involvement of F (integration host factor) was suggested by the similarity of the nucleotide sequence to the Nifa / Ntra-dependent promoter PnifH, and the analysis with DNase 1 identified the binding site and distribution of IHF and XylR in the Pu promoter. There is.

According to the studies to date, it is being recognized in eukaryotes and prokaryotes that transcription initiation is triggered by DNA bending upstream of a positive control promoter. Transcription may also be activated by the naturally occurring bending of DNA in the absence of a binding protein. As a result of analyzing the Pu sequence of this TOL plasmid, a fairly sharp bend was found, and it has been elucidated that it is a site where XylR acts (Fig. 3).

The method utilizing bioluminescence as a detection index, especially luciferase, has the advantages of high sensitivity, rapidity, and easy nondestructive measurement.

By the way, firefly luminescence is the best known bioluminescence, but some microorganisms have luciferase in their cells. These microorganisms are generically called luminescent bacteria, and the luminescent reaction involves various regeneration functions of coenzymes and physiological functions as cells. For this reason, the development of analytical methods using the luminescence intensity of luminescent microorganisms as an index has been actively conducted.

[0017] Regarding luminescent microorganisms, researches on luciferase and luciferin have been conducted in more detail than those of other luminescent organisms. In particular, Vibrio harveyi luciferase has been studied at the molecular level, including its gene sequence. The luciferase, saturated long-chain aliphatic aldehydes such as tetradecanal, reduced FMN (FMNH ₂₎ and oxygen are required for the luminescence reaction of V. harveyi. The luciferase of a luminescent microorganism has a similar molecular weight regardless of its origin and is composed of two different subunits α and β. In V. harveyi luciferase, the α and β subunits have molecular weights of 42,000 and 37,000, respectively, and are simple proteins containing no other components.

The development of the above-mentioned analytical method using the luminescence intensity of the luminescent microorganism as an index has been rapidly progressing together with the progress of the photon counting method. It has already been reported that the dissolved oxygen concentration is measured by monitoring the change in luminescence intensity of luminescent microorganisms, the measurement of hydrogen peroxide using the catalase activity of luminescent bacteria, and the like. Also, luminescent microorganisms
By immobilizing Photobacterium phosphoreum on the membrane,
It is possible to quantify sugar. It is considered that this is because the addition of sugar activates the coenzyme regeneration system such as NAD (P) H-FMN reductase that reduces FMN possessed by luminescent microorganisms. Studies have also been conducted to detect toxic substances by utilizing the decrease in luminescence intensity of luminescent microorganisms. Bulich
Et al. Use Photobacterium leiognathi to measure environmental pollutants. As in this example, researches for detecting various toxic substances from the decrease of luminescence intensity are being actively conducted.

As described above, it is possible to monitor environmental pollutants by using luminescent microorganisms. However, the limitation of this monitoring system was that only luminescent microorganisms could be used.

By the way, the recent development of genetic engineering technology has succeeded in cloning the enzyme luciferase which catalyzes the bioluminescence. In fact, luciferase expression derived from luminescent microorganisms including firefly has been confirmed as a gene activity probe in animal and plant cells, microorganisms, and yeast. Therefore, it is considered that by expressing luciferase in Escherichia coli whose properties such as metabolic system have been elucidated in detail, a monitoring system in which bioluminescence can be used as an index can be systematically constructed.

[0021] Korpela et al. Of Finland, V. harveyi
We have succeeded in constructing luminescent E. coli by cloning the luciferase gene derived from E. coli. (Korpe
la, M., Mantsala, P., Lilius, E.-M., and Karp, M., JB
iolumi. Chemilume., 4 , 551 (1989)) Furthermore, since this luminescent E. coli shows a decrease in luminescence intensity due to the addition of cadmium ions, it is expected to be applied to the monitoring of environmental pollutants (Seiji Nishiguchi). Graduation thesis (199
0)).

Although it is different from the direct monitoring of environmental pollutants, a study of measuring the number of Escherichia coli in soil using such luminescent Escherichia coli has been recently reported.

As described above, it has been clarified that various monitoring can be carried out by introducing luciferase into a host microorganism such as Escherichia coli in which gene analysis has been advanced. However, in the above examples, the luciferase expression level per cell does not depend on the substance to be monitored.

Therefore, for more advanced monitoring, it is conceivable to clone the luciferase gene under a promoter sequence sensitive to the target substance. To date, only one such study has been reported. King et al., NA from Pseudomonas
We have reported naphthalene monitoring using H7 plasmid.

[0025]

[Problems to be Solved by the Invention]

However, in this report, since the luciferase expression control system is dispersed in a plurality of regions, establishment of a simpler system is desired from an engineering point of view.

Therefore, an object of the present invention is to provide a method capable of specifying an organic compound with a simpler system.

[0028]

The method for identifying an organic compound of the present invention is a method for identifying an organic compound in a sample, which comprises introducing a luciferase gene below a promoter of a degrading plasmid that acts on the organic compound. Escherichia coli transformed with the obtained luciferase-expressing plasmid is dispersed in the sample, and the organic compound is specified by the luminescence state at that time.

[0029]

[Effect] Escherichia coli transformed with the luciferase-expressing plasmid of the present invention (see FIG. 5) acts on the organic compound which is an indicator substance, and acts on it at the same time as it expresses luciferase and emits light. . Since the amount of emitted light changes depending on the type and amount of the indicator substance, by measuring the amount of emitted light, the type and abundance of the indicator substance, that is, the organic compound can be specified.

[0030]

DETAILED DESCRIPTION OF THE INVENTION As an example of constructing a plasmid using luminescence measurement as an index, a novel plasmid in which the firefly luciferase gene is cloned under the control of the control region of the TOL plasmid is designed and constructed. For that purpose, the presence of an expression vector system that enables the reconstitution of the control region is essential. Here, the plasmid pTS301 (Fig. 6) was used as an artificial expression vector system in which the control region of the TOL plasmid was reconstructed.
Was used. This plasmid carries the promoters Ps of xylR and xylS (Fig. 7), which are the regulatory regions of the TOL plasmid, and the indicator enzyme gene xylE (catechol 2,3-dioxygenase; C230).

The plasmid pTS301 contains pTS1144 and pTS174.
The Hpal fragment containing the xylR gene was inserted. pTS3
The origin of each area of 01 is as follows. Region A: Rlb679 with Tn1 transferred near oriV of RSF1010
It comes from A. I have entered the location of oriV but it has not been confirmed. Since the Hpal fragment containing xylR was inserted into the Eco RI site, the Eco RI site does not exist. Region b: Xhol fragment containing xylE in Xhol of pACYC177 (2.2k
After insertion of b), the xylS and xylR promoters were inserted into the BamHI, so there is no BamHI site. Also at this time
A part of pACYC177 is deleted.

Examples of the luciferase bioluminescent substance of firefly (Photinus pyralis) include, for example, firefly (Photinus pyralis).
alis) luciferase is known, and this firefly luciferase is also used in this example. This firefly luciferase has an excellent quantum efficiency (0.88 ± 0.
25) and the whole nucleotide sequence of the luciferase gene has been determined, which is convenient for gene design.

Firefly luciferase is an enzyme having a molecular weight of 62,000, and catalyzes a luminescence reaction by oxidizing firefly luciferin in the presence of ATP, the enzyme and Mg ²⁺ (FIG. 4). The reaction formula is shown below. luciferin + ATP + luciferase → luciferase ・ lucife
ryl-AMP + PPi Mg ²⁺ luciferase ・ luciferyl-AMP + O ₂ → luciferase + oxyl
uciferin + AMP + CO ₂ + hν (560nm)

In order to increase the active expression of the firefly luciferase gene due to the presence of BTXs, it is necessary to clone luciferase under the promoter Ps of the xylS region whose expression is controlled by the XylR protein. Therefore, here, in consideration of the restriction map, the 13.8 kb generated by treating the plasmid pTS301 with the restriction enzyme KpnI.
We decided to use fragments. The KpnI fragment (13.8 Kb) of pTS301 contains the target xylR and xylS promoters.
The replication regions of Ps and RSF1010 are present, and a plasmid capable of autonomous replication can be constructed.

Therefore, in order to clone firefly luciferase into the Ps downstream of the KpnI fragment of pTS301, the following three methods can be considered. Insert firefly luciferase into KpnI. It is partially digested with HpaI and firefly luciferase is inserted downstream of Ps. After checking whether SstII is downstream of xylR, insert it into SstII if it is not present.

Construction of Actual Plasmid Here, as an example, a plasmid was constructed by the above method.

The following reagents were used to construct the reagent plasmids. The restriction enzyme is Takara Shuzo Co., Ltd. or Bethesda Research Laboratorie.
Purchased from s, Inc. Bacterial Alkaline Phosphatase
Was a product of Toyobo Co., Ltd. The T4 DNA Ligase used was purchased from Toyobo Co., Ltd., and the DNA Ligation Kit purchased from Takara Shuzo Co., Ltd. was used. All other reagents were of special grade.

Strain and plasmid Escherichia coli JM109 was purchased from Takara Shuzo Co., Ltd. Also E. coli
20SO and HB101 were used from the Department of Microbiology, Yamaguchi University School of Medicine.

The plasmid pT3 / T7-luc encoding the firefly luciferase gene was purchased from Toyobo Co., Ltd. The plasmids pTS301 and pTH3 were obtained from the Department of Microbiology, Yamaguchi University School of Medicine. E.coli JM109 recAl endAl gyrA96 thi-1 supE44 relAl λ - Δ (lac-proAB) [F'traD36 proAB lacI ^q Z ΔM15] ^{mcrA - mcrB + HB101 F - hsds20} reaA13 ara14 proA2 lacY1 galK2 rpsL20 x15 mtll supE44 mcrA + mcrB ^{- 20SO thi lac mal mtl ara xyl} rpsL Plasmids pT3 / T7-luc luciferase expression vector pTS301 XylR + xylS promoter Amp r PTH3 Km r

Construction of plasmid In order to introduce the firefly luciferase gene (1.9 kb) into the KpnI fragment (13.8 kb) of the plasmid pTS301, it is conceivable to blunt the ends of each gene fragment and then ligate each of them. . This method uses T4 DNA Polymerase 5 '
This is a method that utilizes both having a 3'-polymerase activity and a 3 '->5' exonuclease activity, and is a highly versatile method that is not affected by the nucleotide sequence of the cleavage terminal. However, the method of blunting is less efficient than the ligation reaction between sticky ends, and thus may be said to cause some difficulties in obtaining the desired plasmid. Therefore, Mult was added to the KpnI-cut end of the plasmid pTS301.
It is preferable to first construct a plasmid into which i Cloning Site (MCS) has been introduced, and clone the firefly luciferase gene into this. MCS was introduced as follows.

Introduction of Multi Cloning Site (MCS) (FIG. 8) After cutting the plasmid pTS301 with the restriction enzyme KpnI,
The 13.8 kb fragment was recovered and purified. Similarly, plasmid pTH3
Was treated with the restriction enzyme KpnI to obtain a 1.3 kb fragment. E. coli HB101 was transformed with this new plasmid by ligating them with T4 DNA Ligase according to a conventional method. For screening the target strain, 13.8 of plasmid pTS301 was selected.
The kb fragment confers ampicillin resistance to the 1.3 kb plasmid pTH.
It was taken advantage of that the fragment has kanamycin resistance.

At the BamHI site in the MCS site of the MCS-introduced plasmid constructed in the step of introducing the firefly luciferase gene into MCS (FIG. 9), the firefly luciferase gene was introduced.
The target plasmid was constructed by introducing (1.9 kb).
The method is described below.

The MCS-introduced plasmid was digested with the restriction enzyme BamHI, and the 13.8 kb fragment was recovered and purified. This 13.8kb
The 5'-terminal phosphate group of the fragment was changed to Bacterial Alkaline Phospha
By removing with tase to generate a hydroxyl group, recyclization of the vector was prevented. Then, the plasmid pT3 / T7-luc was digested with the restriction enzymes BamHI and KhoI to recover a 1.9 kb BamHI fragment. This fragment encodes the entire base sequence of firefly luciferase. Therefore, these fragments were ligated with the DNA Ligation Kit to construct the target plasmid. Escherichia coli HB101 was transformed with this plasmid. Ampicillin resistance was used for the screening.

Experimental Results Introduction of MCS The plasmid into which MCS was introduced was treated with a restriction enzyme and confirmed by a known genetic map.
The restriction enzyme BglII is contained in the KpnI fragment of plasmid pTS301.
There are four cutting sites. Also, the plasmid pT
It is known that there is one BglII site in the KpnI fragment of H3. Therefore, restriction enzyme treatment with BglII was performed on the plasmid into which MCS had been introduced.

As a result, the introduction of MCS was confirmed, and at the same time the insertion direction could be determined (FIG. 10). These plasmids are designated as pTSN9 and pTSN12.

Introduction of Firefly Luciferase Gene into MCS The plasmid pTSN9 or pTSN12 was treated with BamHI, and the firefly luciferase gene was introduced into the plasmid to construct the target plasmid. When analyzing the plasmid structure, restriction enzymes KpnI and SacI were used at the same time. This makes it possible to confirm the position of the SacI site that was considered to exist in the KpnI fragment of plasmid pTS301.

Furthermore, by using the SacI site which is clearly present in the firefly luciferase gene,
It is also possible to determine the introduction direction of luciferase.

As a result, it was found that three types of plasmids were newly obtained, including the presence or absence of the luciferase gene insertion (FIG. 11). Therefore, these plasmids were designated as pTSN 302, pTSN304 and pTSN316, respectively. pTSN302
Is a plasmid in which the firefly luciferase gene has not been inserted. In addition, pTSN304 has the luciferase gene inserted in the opposite direction to Ps.
Is the desired plasmid. From the above, the following novel plasmid was obtained.

Vector system plasmid having MCS pTSN9 XylR ⁺ xylS promoter (Ps) Amp ^r Km ^r pTSN12 XylR ⁺ xylS promoter (Ps) Amp ^r Km ^r A plasmid obtained by introducing a firefly luciferase gene (luc) into MCS pTSN304 XylR ⁺ xylS promoter (Ps) luc Amp ^r pTSN316 XylR ⁺ xylS promoter (Ps) luc Amp ^r Recyclization of KpnI fragment of plasmid pTS301 pTSN301 XylR ⁺ xylS promoter (Ps) Amp ^r

Basic characteristics as marker microorganisms E. coli transformed with the plasmid pTSN316
Detection of organic compounds Hereinafter, specific experimental examples of luminescence monitoring of BTXs using Escherichia coli HB101 (pTSN316) transformed with the above plasmid pTS301 will be described.

The firefly luciferase gene used as an indicator enzyme in this experiment was cloned from North American firefly Photinus pyralis by DeLuca et al. Of the University of California. In the plasmid pT3 / T7-luc, the gene region encoding firefly luciferase is present in the BamHI fragment consisting of 1.9 kb. Also, this area is 55
It has an open reading frame (ORF) of 1650 base pairs that enables total synthesis of firefly luciferase consisting of 0 amino acid residues.

As described above, the luminescent reaction of firefly is
The enzyme luciferase and the substrate luciferin, ATP,
Mg ²⁺ and O ₂ are required. By the way, when firefly luciferase is expressed in Escherichia coli, a problem occurs in the supply of these substrates. For the purpose of luminescence monitoring of environmental pollutants, the biggest difficulty in using a molecularly bred luminescent microorganism is that the production system of luciferin as a substrate does not exist in the host cell. Luminescent bacteria V.harv
At eyi, a group of enzymes that synthesize a saturated long-chain aldehyde that serves as a substrate has been cloned together with the luciferase gene, and the bioluminescence system can be reconstructed relatively easily. On the other hand, in the firefly luminescence system, the luciferin synthase group has not been identified yet, and the firefly luciferin must be supplied from the outside of the cells.

Further, as described above, the regulatory gene xyl product R (XylR protein) of the TOL plasmid exhibits affinity for a specific aromatic compound in an organic compound, and controls gene expression in the TOL plasmid. . Among aromatic compounds, benzene, toluene, xylene, etc. are used industrially on a large scale as environmental pollutants B
TXs are the most versatile targets. The actual condition of the monitoring system depending on the concentration or type of these benzene derivatives by the molecularly-bred microorganism will be described later.

First, the luminescence of Escherichia coli HB101 (pTSN316) transformed with the above constructed plasmid in the presence of the inducer was confirmed, and then the luminescent response to the inducer concentration and the inducer species was examined. In addition, an experiment was conducted to determine whether the luminescence produced by the cells corresponds to the amount of luciferase produced inside the cells.

Experimental Method Reagent D-(-)-luciferin was purchased from Boehringer Mannheim GmbH. Yeast Extrat and Bacto Trypton used were manufactured by Difco. The ampicillin sodium used was that of Wako Pure Chemical Industries, Ltd. Aromatic compounds such as benzene, toluene, o-, m-, and p-xylene used as inducers were all of special grade. All other reagents were of special grade.

Culture method

For culturing E. coli HB101, L broth (1% t
rypton 0.5% yeast extract, 0.5% NaCl, pH 7.4) was used, and ampicillin was added to the medium to a final concentration of 100 μg / ml. Culture temperature is 30 ° C, shaking speed is 1 minute
I set it to 40 rpm. The precultured bacterial solution (2% concentration) was inoculated into the medium, and the culture was started at this point.

For the induction of luciferase, Nakazawa
These methods were referenced. That is, when using benzene or toluene as the inducer, a 200 ml Erlenmeyer flask containing 50 ml of L broth was sealed and placed in this flask.
A test tube (diameter 10 mm, length 100 mm) containing 0.2 ml of each aromatic compound was fixed. As a result, the aromatic compound is dissolved in the medium while maintaining a gas-liquid equilibrium relationship. In addition, m-methylbenzyl alcohol (m-MBA)
Dissolved directly in L broth.

Measuring Device and Method Measuring Device A photon counting system was used for luminescence measurement. The outline of the measuring device is shown in FIG. As shown in the figure, the measuring device is composed of a dark box 1, a photomultiplier tube (photomultiplier tube) 2, a photon counter 3, a selling unit 4, a digital storage oscilloscope 5, and an XY recorder 6.
A constant temperature cell holder is installed in the dark box, and luminescence measurement can be performed at a constant condition of 30 ° C.

Photomultiplier tube (R464),
The photon counter (C1230) was manufactured by Hamamatsu Photonics Co., Ltd., the digital storage oscilloscope (DS-6612C) was manufactured by Iwasaki Tsushinki Co., Ltd., and the XY recorder (WX2400) was manufactured by Graphtec Co., Ltd. An ultraviolet-visible spectrophotometer (JASCO Ubest-30) was used to measure the bacterial density of E. coli.

Luminescence measurement by E. coli E. coli cultured in L broth was sampled after a predetermined time, and the bacterial density was measured by an ultraviolet-visible spectrophotometer. (A660).
After centrifuging 1 ml of these cells, 1 ml of STE buffer (100 mM N
Wash twice with aCl, 10mM Tris-Cl pH7.8, 1mM EDTA (4
℃). The collected cells are suspended in 1.8 ml of the same buffer and incubated at 30 ° C for 5 minutes. To this, add 0.2 ml of D-(-)-luciferin solution to a final concentration of 0.1 mM, and make the volume in the cell 2.0 ml. At this time, the change in the liquid amount in front of the light receiving window of the photomultiplier tube can be ignored. The reaction start time was defined as the time when the D-(-)-luciferin solution was added, and the amount of luminescence after a certain period of time is evaluated by the number of photons (cps) per second.

Measurement of luciferase activity in soluble fraction DeLu was used for the preparation of soluble fraction and measurement of luciferase activity.
Improvements were made to the method of ca et al. The method will be described below.

After measuring the bacterial density (A660) of E. coli, culture medium 1
Centrifuge the cells in ml and remove the supernatant by decantation.
Add 0.8 ml of lysis buffer (10 mM Tris-Cl, pH8.0 / 1.0
The cells were suspended in mM EDTA / lysozyme (1 mg / ml), left for 15 minutes on ice, and then frozen at -80 ° C. Then melt at 25 ° C, 15
The precipitate was removed by centrifugation at 000 rpm for 15 minutes. The supernatant was used as the soluble fraction of luminescent E. coli.

0.2 ml of the above soluble fraction was used for measuring luciferase activity. Soluble fraction 0.2 ml to 25 mM
Dilute with 1.2 ml of glycylglycine buffer solution (pH 7.8), and add 0.2 ml of 20 mM ATP solution (pH 7.8). Substrate mixture (0.1 mM D-(-)-luciferin / 5 mM MgCl ₂ ) was added to this solution.
The luminescence reaction was started by adding 0.4 ml of / 25 mM glycylglycine). It was decided to evaluate the enzyme activity based on the height of the peak observed immediately after the start of the reaction.

Results Time-dependent changes in luminescence m-xylene was used as a typical inducer acting on the xylR and xylS gene regions of the TOL plasmid. At this time, it was found from FIG. 14 that Escherichia coli cultivated under the condition of being brought into contact with the gas phase saturated with m-xylene was hardly affected by the growth rate as compared with Escherichia coli cultivated in the non-induced state. .

When the change in the luminescence amount at each growth stage was measured, the results shown in FIGS. 15, 16 and 17 were obtained.

FIGS. 18 and 19 show the amount of luminescence of Escherichia coli sampled at each growth stage after 5 minutes from the start of the luminescence reaction with respect to the culture time and the bacterial density. When m-xylene was used as the inducer, the luminescence was observed about 10 times higher than the luminescence from the cells cultured in the non-induced state. Therefore, plasmid pTSN31 designed and constructed as described above.
Aromatic compounds can be detected by Escherichia coli HB101 transformed with 6.

The change in luminescence of bacterial cells when m-xylene was used as a correlative inducer of luciferase activity was described above. By the way, in Gram-negative bacteria including Escherichia coli, it is difficult for a hydrophobic compound or a compound having a large molecular weight to be taken up into the cells by the outer membrane (OM) as a barrier.
On the other hand, it is said that the hydrophilic compound is taken into the microbial cells relatively easily. Although these are phenomena caused by the hydrophobicity of the biological membrane, as shown in FIG. 16, the delay in the response rate of the luminescence reaction is due to the poor membrane permeability of luciferin, that is, the hydrophobicity of luciferin. Therefore, in this experiment, the cells were washed with a buffer solution containing EDTA before adding luciferin. This allows the E. coli lipopolysaccharide membrane (L
It promoted the intracellular permeation of hydrophobic compounds by causing the destruction of PS) and increasing the hydrophobicity of the outer membrane.

Here, by examining the correlation between the luminescence from the bacterial cells and the amount of luciferase inside the bacterial cells,
We examined the luciferase induction of environmental pollutants in E. coli. The results are shown in FIGS. 20 and 21. here,
The amount of intracellular luciferase used that corresponds to the height of the first peak in FIG.

From the above results, it was clarified that the luminescence by the bacterial cells correlates well with the intracellular luciferase amount. Further, in the evaluation of the luminescence amount using the bacterial cells, the luminescence amount 5 minutes after the start of the luminescence reaction was set as the reference value.

Luminescence vs. Inducer Concentration Here, m-methylbenzyl alcohol was used as the inducer.
(m-MBA) was used. m-MBA has been shown to act on the TOL plasmid regulatory genes xylR and xyls,
Since it is soluble in an aqueous system, it is considered that the concentration in the medium and the amount of luminescence can be easily quantified. However, m-MB directly in the medium
The addition of A may affect the growth of E. coli. It is generally said that aromatic compounds such as m-MBA destroy the outer membrane of microorganisms. Therefore, it is considered that some change occurs in the membrane permeability of luciferin. Therefore, here, the luminescence by the bacterial cells at each growth stage was measured, and at the same time, the correlation with the luminescence by the soluble fraction was examined. The results are shown in FIGS. 22, 23, 24 and 25.

From FIG. 22, it was revealed that the presence of m-MBA suppressed the growth of E. coli depending on the concentration thereof. Table 2 shows the growth rate of Escherichia coli for each set concentration.

[0073]

[Table 2]

Further, it was found from FIG. 25 that the luminescence of the cells increased with the concentration of m-MBA. Furthermore, it was revealed from FIG. 25 that there is a correlation between the luminescence by the bacterial cells and the luminescence by the soluble fraction thereof. From the above results, it is possible to measure the concentration of the benzene derivative existing in the environment by using Escherichia coli HB101 (pTSN316), which is a microorganism bred in this experiment.

Search for Inducer Species Next, another aromatic compound that could be an inducer of luciferase expression in Escherichia coli HB101 (pTSN316) was searched. This search is also an affinity evaluation of xylR protein and aromatic compounds (see FIG. 3). The selection criterion for the inducer is a report on the evaluation of transcriptional activity of operon 1 and operon 2 by xylR protein (abril, M.-A., Michan, C., Timmis,
KN and Ramos, JL, J. Bacteriol., 171 , 6782 (1
989)). According to this document, the structure of an aromatic compound that interacts with the xylR protein requires that one -CH ₃ or -CH ₂ OH group be attached to the benzene ring. On the other hand, an alkyl group or a chloro group may be present on the carbons at the -o, m-, p-positions. The aromatic compound recognition site of the xylR protein has symmetry, and a substituent may be present at the o-position or m-position. Only when the chloro group is introduced at the carbon at the p-position, introduction of the -CHO group at the 1-position is allowed. There is a principle. However, the issue here is the promoter region of operon 1 and operon 2, namely P
Induction of transcriptional activity by xylR protein for u and Pm. In the plasmid pTSN316 constructed above, the firefly luciferase gene was cloned downstream of the promoter Ps of xyls, so a subtle difference in the gene sequence to which the xylR protein binds may appear as a difference in transcription activity. Therefore, in this section, we decided to reexamine the above-mentioned principles. Therefore, we decided to investigate the possibility of the following aromatic compounds as inducers, and first performed luminescence measurements on the isomer structure of m-xylene, which is a strong inducer of TOL plasmid.

Measurement Aromatic compounds Benzene, toluene, ethylbenzene o-, m-, p-xylene o-, m-, p-ethyltoluene o-, m-, p-chlorotoluene 1,2,3-, 1, 2,4-, 1,3,5-Trimethylbenzenebenzaldehyde

Search for inducer species Induction with o-, m-, p-xylene The growth curves in the presence of each inducer were as shown in FIG. 26. From this figure, there was no difference in the growth of E. coli.

Next, the time-dependent change in luminescence at each growth stage was observed. The results are shown in FIGS. 27, 28 and 29. As a result, it was shown that the luminescence-inducing effect of xylene differs depending on the growth stage. That is, it was revealed that the emission from m-xylene was larger than the emission from other isomeric structures from the middle to the late logarithmic growth phase. Then, the amount of luminescence with respect to the bacterial density was plotted in FIG.
As a result, the induction of luminescence by each xylene isomer was slightly different, and in particular, the difference in luminescence behavior depending on the growth stage of Escherichia coli could be observed. In Escherichia coli HB101 (pTSN316), the luciferase activity is the regulatory gene product xy of the TOL plasmid.
It is regulated by the lR protein. Therefore, if the luciferase production is controlled only by the xylR protein, this luminescence by Escherichia coli is considered to reflect the production amount of the xylR protein itself. It is also considered to reflect the relationship between auxiliary factors such as IHF, as mentioned earlier.

Next, relative evaluation of luminescence of Escherichia coli HB101 (pTSN316) cultured in the absence of an inducer was carried out for each isomer of xylene. The result is shown in FIG. FIG. 31 means that the relative amount of light emitted from the bacterial cells by each inducer hardly depends on the reaction time.

Therefore, the subsequent luminescence measurement was carried out by using Escherichia coli HB101 (pTSN) cultured under conditions containing these organic compounds in the gas phase.
316) was sampled when the bacterial density was A ₆₆₀ = 1.0. Further, the evaluation of the luminescence amount was made by comparing the luminescence amount 5 minutes after the initiation of the luminescence reaction with the luminescence amount from the non-induced cells.

Search for inducer species a Toluene, ethylbenzene, benzaldehyde b benzene, o-, m-, p-ethyltoluene co-, m-, p-chlorotoluene d 1,2,3-, 1,2, 4-, 1,3,5-trimethylbenzene

Luminescence was measured for the above 13 kinds of aromatic compounds, and the amount of luminescence by the fungus body was relatively evaluated for each compound. a The results are shown in FIGS. 32, 33 b 34, 35 c 36, 37 d 38 and 39.

Table 3 shows the results of relative evaluation of the amount of emitted light.

[0084]

[Table 3]

From these results, it is clear that the above-mentioned principle can be applied to an aromatic compound having a structure which can be recognized by the xylR protein (has an affinity).

The fluctuation of the relative luminescence amount due to the bacterial cells is shown in FIG.
As shown in FIG. 3, FIG. 35, FIG. 37 and FIG. Therefore, a rapid and non-destructive luminescence measurement of environmental pollutants was suggested by E. coli HB101 (pTSN316).

As described above, by using Escherichia coli transformed with a plasmid whose luminescence measurement is used as an index, it becomes possible to detect BTXs which are environmental pollutants. That is, Escherichia coli HB101 transformed with the plasmid pTSN316 in which the firefly luciferase gene was cloned under the control of the promoter (Ps) of the regulatory gene region xyls of the TOL plasmid that has a complete degrading enzyme system for xylene was used, and the specific aromatic compound type and concentration were It has been revealed that luminescence is generated in response to.

[0088]

[Effects] Escherichia coli HB101 (pTSN316) subjected to molecular breeding can be used for luminescence monitoring of organic compounds such as aromatic compounds as environmental pollutants. Since the amount of emitted light by the constructed microorganism increases according to the concentration of the organic compound, the amount of the inducer present in the environment can be measured by measuring the amount of emitted light. Luminescence by the bacterial cells corresponds to the amount of luciferase produced. The structure of the aromatic compound, especially the type and position of the functional group, controls the induction of luciferase activity. This is thought to be due to the specificity of the xylR protein, which is a regulatory gene xylR product, for recognizing organic compounds.

As described above, since the constructed luminescent Escherichia coli emits luminescence depending on the type and concentration of the benzene derivative, it is possible to selectively detect environmental pollutants by molecular breeding of microorganisms using bioluminescence as an index. it can. At this time, in addition to the TOL plasmid, the corresponding various degradative plasmids can be used in the same manner to detect the corresponding various degrading organic compounds.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a degradation pathway by a TOL plasmid.

FIG. 2 is a diagram showing a gene control mechanism in a TOL plasmid.

FIG. 3 is a diagram showing a transcription activation model by Xy1R protein in the presence of m-xylene.

FIG. 4 is a diagram showing a luciferin-luciferase reaction mechanism (firefly).

FIG. 5 is a diagram showing the concept of luminescent measurement marker microorganisms.

FIG. 6 shows the plasmid pTS301.

FIG. 7 shows the nucleotide sequences of xy1R and xy1S promoter regions.

FIG. 8 is a diagram showing the introduction of a Multi Cloning Site (MCS).

FIG. 9 is a diagram showing the introduction of firefly luciferase into MCS.

FIG. 10 is a drawing-substitute copy showing an analysis by agarose gel electrophoresis.

FIG. 11 is a drawing-substituting photograph showing analysis by agarose gel electrophoresis.

FIG. 12 is a diagram showing a restriction enzyme map of each plasmid.

FIG. 13 is a schematic view showing a measuring apparatus for carrying out the method of the present invention.

FIG. 14: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
It is a graph which shows the growth curve of 6).

FIG. 15: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
It is a graph which shows the growth curve (logarithmic initial stage) of 6).

FIG. 16: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
It is a graph which shows the growth curve (mid logarithm) of 6).

FIG. 17: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
It is a graph which shows the growth curve (late log phase) of 6).

FIG. 18: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
It is a graph which shows the light emission behavior of 6) (Luciferin dripping 5min. Progress).

FIG. 19: Escherichia coli HB101 (pTSN31 in the presence of m-Xylene
FIG. 6 is a graph showing the number of cells of 6) and its luminescence behavior (after 5 minutes of Luciferin dropping).

FIG. 20 is a graph showing an L-Lase reaction.

FIG. 21. Escherichia coli HB101 (pTSN31 in the presence of m-Xylene.
6) and its lysate emission behavior (Luciferin drop 5 mi
It is a graph figure which shows n.

FIG. 22 is a graph showing the induction by m-MBA and the number of Escherichia coli HB101 (pTSN316) cells.

FIG. 23 is a graph showing induction by m-MBA and luminescence behavior of Escherichia coli HB101 (pTSN316) (after 5 minutes of dropping Luciferin).

FIG. 24 is a graph showing induction by m-MBA and luminescence behavior of Escherichia coli HB101 (pTSN316) and its lysate (after 5 minutes of Luciferin dropping).

FIG. 25 is a graph showing the m-MBA concentration and the luminescence behavior of Escherichia coli HB101 (pTSN316) (after 5 minutes of dropping Luciferin).

FIG. 26: Escherichia coli HB with o-Xylene, m-Xylene, p-Xylene
FIG. 6 is a graph showing induction of 101 (pTSN316).

FIG. 27 is a graph showing induction by o-Xylene, m-Xylene, p-Xylene and luminescence behavior of Escherichia coli HB101 (pTSN316).

FIG. 28 is a graph showing induction by o-Xylene, m-Xylene, p-Xylene and luminescence behavior of Escherichia coli HB101 (pTSN316).

FIG. 29 is a graph showing induction by o-Xylene, m-Xylene, p-Xylene and luminescence behavior of Escherichia coli HB101 (pTSN316).

FIG. 30 is a graph showing induction by o-Xylene, m-Xylene, p-Xylene and proliferation and luminescence behavior of Escherichia coli HB101 (pTSN316).

FIG. 31: E. coli HB with o-Xylene, m-Xylene, p-Xylene
FIG. 4 is a graph showing the luminescence induction of 101 (pTSN316) and its relative intensity.

FIG. 32 is a graph showing the luminescence behavior of Escherichia coli HB101 (pTSN316) by Toluene, Ethylbenzene and Benzaldehyde.

FIG. 33 is a graph showing luminescence induction of Escherichia coli HB101 (pTSN316) by Toluene, Ethylbenzene, and Benzaldehyde and its relative intensity.

FIG. 34 is a graph showing luminescence induction of Escherichia coli HB101 (pTSN316) by Bezene, o-Ethylbenzene, m-Ethylbenzene and p-Ethylbenzene.

FIG. 35 is a graph showing luminescence induction of Escherichia coli HB101 (pTSN316) by Bezene, o-Ethylbenzene, m-Ethylbenzene and p-Ethylbenzene and its relative intensity.

FIG. 36: o-Chlorotoluene, m-Chlorotoluene and p-
FIG. 3 is a graph showing luminescence induction of Escherichia coli HB101 (pTSN316) by Chlorotoluene.

FIG. 37: o-Chlorotoluene, m-Chlorotoluene and p-
FIG. 3 is a graph showing luminescence induction of Escherichia coli HB101 (pTSN316) by Chlorotoluene and its relative intensity.

Figure 38: 1,2,3-Trimethylbenzene, 1,2,4-Trimethylbe
Escherichia coli HB101 by nzene and 1,3,5-Trimethylbenzene
It is a graph which shows the light emission induction of (pTSN316).

Figure 39: 1,2,3-Trimethylbenzene, 1,2,4-Trimethylbe
Escherichia coli HB101 by nzene and 1,3,5-Trimethylbenzene
FIG. 4 is a graph showing the luminescence induction of (pTSN316) and its relative intensity.

[Explanation of symbols]

 1 Dark Box 2 Photo Multiplier Tube 3 Photo Counter 4 Suring Unit 5 Digital Storage Oscilloscope 6 XY Recorder

Front page continuation (72) Inventor Eri Kobata 1-18, Minami 18-jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido (72) Inventor Seiji Nishiguchi 4-6-30 Shioya, Wakayama, Wakayama Prefecture

Claims (1)

[Claims] 1. A method for identifying an organic compound in a sample, which comprises transforming E. coli transformed with a luciferase-expressing plasmid obtained by introducing a luciferase gene below a promoter of a degradation plasmid that acts on the organic compound,
Dispersed in the sample, depending on the state of light emission at that time,
A method for identifying an organic compound for identifying an organic compound.