JP2941210B2 - Microbial identification, detection and monitoring method using aromatic hydroxylase dioxygenase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention detects, at the genetic level, bacteria that play an important role in various industries such as medicine, food and chemistry, and environmental protection such as biological decomposition of pollutants and water treatment. The present invention relates to a method, a method for identifying these bacteria, and a method for monitoring the activity and growth of these bacteria. More specifically, the present invention relates to a method for detecting and identifying an aromatic-degrading bacterium that degrades an aromatic compound, and a method for monitoring the growth thereof.

[0002]

2. Description of the Related Art In order to effectively cope with environmental pollution caused by various contaminants, the method of decomposing the contaminants and the degree of decomposition have been advanced. It is necessary to know whether or not to take effective measures. Traditionally, biologically degraded sources of pollution,
For example, when the environment is contaminated by crude oil, etc., a method for analyzing the concentration of various hydrocarbons contained in crude oil is used to understand the degree of biological degradation of crude oil, etc., which is the pollution source. I have been.

[0003]

However, the method of analyzing various hydrocarbons in crude oil has a problem that it is difficult to obtain an accurate analysis result despite the complicated operation and the time required. .

[0004] In addition, since the analysis of hydrocarbons in crude oil requires a complicated apparatus, there are few cases where analysis equipment is installed at a site where environmental purification is performed. Therefore, in most cases, it is necessary to collect and store analytical samples and transport them to a location where they can be analyzed. However, these samples deteriorated during storage and transportation, and the obtained results sometimes lacked reproducibility and sometimes did not reflect the state of biodegradation of crude oil at the contamination site.

[0005] Hydrocarbons contained in crude oil and the like are roughly classified into saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes. Among these components, aromatic hydrocarbons,
Since both resin and asphaltene contain aromatic compounds, they are less susceptible to degradation by microorganisms. For this reason, the concentration of these aromatic compounds in crude oil can be used as an index of how much contaminants such as crude oil have been decomposed. However, it has been difficult to grasp what components are decomposed in the above contaminants such as crude oil and what components remain without being decomposed.

As described above, since it is difficult to analyze aromatic hydrocarbon compounds in crude oil, in order to know the state of biological degradation of crude oil, bacteria that degrade hydrocarbons in crude oil were identified. It was conceived to adopt a method of doing so. However, conventional methods for identifying bacteria by biochemical tests based on sugar assimilation and the like do not always provide accurate results despite many test items and complicated and time-consuming operations. There was no problem.

In addition, in order to detect bacteria having a specific function with high accuracy, 16S microorganisms, which have been recently used, have been used.
A method using gene level analysis using the base sequence of rRNA of the above is also conceivable. However, 16S rRNA
Is a gene held by all microorganisms,
It is difficult to detect only a group of microorganisms having a specific metabolic mechanism. Further, in order to perform detection using a gene common to microorganisms such as 16S rRNA, 16S rRNA is required.
It was necessary to perform a complicated operation of amplifying a specific base sequence of RNA by PCR, cloning the amplified fragments one by one, and performing sequencing. In addition, since the base sequence of the 16S rRNA has little difference when compared between the same genera or species, it was necessary to determine a long base sequence using a plurality of primers.

[0008]

In order to solve the above problems, it is necessary to establish a method for easily detecting and identifying microorganisms or bacterial groups having a specific function with high accuracy. The inventors of the present invention have conducted various studies to solve the above-mentioned problems, and as a result, have found that aromatic ring hydroxylase dioxygenase (hereinafter referred to as dioxygenase) commonly present in certain microorganisms, in particular, aromatic degrading bacteria. (Hereinafter abbreviated as oxygenase) was found to have a conserved amino acid sequence. Based on this finding, the present invention has been completed using these sequences.

[0009] That is, in the present invention, the first invention is directed to an amino acid sequence of an aromatic hydroxylase dioxygenase.
Region that is conserved among the microorganisms to be detected.
This is a method for detecting a microorganism using a base sequence encoding a region or a base sequence complementary thereto . In this case, the number of the areas having the preservability may be two.
The amino acid sequence of the region is, for example, one of Cys-Ser
-Tyr-His-Gly-Trp or Cys-Se
An amino acid sequence consisting of r-Phe-His-Gly-Trp, the other being Asn-Trp-Lys-Phe-
It is an amino acid sequence represented by Ala-Ala-Glu .

A second aspect of the present invention is to provide an aromatic ring hydroxylase
Microbes to be detected in the amino acid sequence of cigenase
Base sequence encoding two regions that are conserved between products
Detect microorganisms using a sequence or its complementary base sequence
After that, the amino acid sequence flanked by the two regions is copied.
This is a method for identifying a microorganism using a base sequence to be loaded . Here, the amino acid sequences of the two regions are, for example,
If one is Cys-Ser-Tyr-His-Gly-
Trp or Cys-Ser-Phe-His-Gly
-An amino acid sequence consisting of Trp, the other being Asn-
It is an amino acid sequence represented by Trp-Lys-Phe-Ala-Ala-Glu . Examples of the microorganism include aromatic decomposition bacteria.

A third aspect of the present invention relates to a method for detecting a microbe in the amino acid sequence of an aromatic hydroxylase dioxygenase.
Nucleotide sequence also encodes a region having a conserved among things
By using a nucleotide sequence complementary thereto, Fang contained in crude oil
This is a method of detecting the growth of aromatic degrading bacteria that decompose aromatic compounds and monitoring the biological decomposition of crude oil using the detected amount as an index. Here, the area having the preservability is
May be two, and in this case the amino acids of the two regions
The sequence is, for example, one of Cys-Ser-Tyr-Hi
s-Gly-Trp or Cys-Ser-Phe-H
an amino acid sequence consisting of is-Gly-Trp, the other being Asn-Trp-Lys-Phe-Ala-Ala
-An amino acid sequence represented by Glu .

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The present invention is a method for detecting a microorganism using a base sequence encoding a specific amino acid sequence present on an aromatic ring hydroxylase dioxygenase gene, which is an enzyme commonly present in certain microorganisms, Further, the present invention is a method for identifying a microorganism detected using the above-mentioned nucleotide sequence.
Furthermore, the present invention detects whether the microorganism having the enzyme is growing in crude oil by using the base sequence,
It is a method to monitor the biological decomposition of crude oil.

Specifically, PCR is carried out using the DNA of the microorganism to be detected using the nucleotide sequence encoding the conserved amino acid sequence present on the dioxygenase gene as a template to obtain a PCR amplification product. This is a method for detecting the above-mentioned microorganisms depending on whether or not the microorganism is obtained.

The aromatic ring hydroxylase dioxygenase is an enzyme that participates in the production of a diol compound by adding an oxygen atom to an aromatic ring, and is also called a diatomic oxygenase. Aromatic hydroxylase dioxygenase is an intramolecular dioxygenase in which one substrate receives two atoms of oxygen in most cases, and requires iron, heme, copper, etc. as cofactors.

The aromatic hydroxylase dioxygenase is widely distributed in aromatic degrading bacteria which degrade aromatic compounds.
It may be present on a chromosome or on a plasmid. The enzyme consists of four units: one large subunit (molecular weight: about 51,000), one small subunit (molecular weight: about 22,000), ferredoxin (molecular weight: about 12,000), and ferredoxin reductase (molecular weight: about 43,000). It is a multi-component enzyme with a molecular weight of about 128,000.

When the amino acid sequences of the aromatic ring hydroxylase dioxygenase genes of a plurality of bacteria were determined and examined, it was found that a universally conserved amino acid sequence exists on the large subunit of this enzyme. Was revealed. These sequences are: Cys- Ser-Tyr- His
-Glv-Trp or Cys- Ser-Phe- Hi
s-Gly-Trp and Asn-Trp-Lys-
It is represented by the amino acid sequence Phe-Ala-Ala- Glu . Highly conserved portions of the above sequences are underlined.

Therefore, the nucleotide sequence encoding these amino acid sequences or the nucleotide sequence complementary thereto is the same as that of PC
It can be used as a primer for amplifying an amino acid sequence flanked by these amino acid sequences by R. Cys-Ser-Tyr-His-Gly-T
rp (hereinafter referred to as amino acid sequence 1) or Cys-S
er-Phe-His-Gly-Trp (hereinafter referred to as amino acid sequence 2) is Asn-Trp-Lys-Phe
The base encoding the amino acid sequences 1 and 2 is located on the upstream side of the amino acid sequence of the large subunit of the aromatic ring hydroxylase dioxygenase, rather than -Ala-Ala-Glu (hereinafter referred to as amino acid sequence 3). The sequence is used as a sense primer, and a base sequence complementary to the base sequence encoding amino acid sequence 3 is used as an antisense primer.

The nucleotide sequences encoding the above amino acid sequences 1 to 3 may be used directly as primers during PCR, or may be used by designing primers based on these. Using these primers, the base sequence encoding the amino acid sequence sandwiched between the
Amplify as fragments. Thereafter, a specific microorganism is detected by detecting a DNA fragment amplified by electrophoresis, DNA hybridization, or the like, or a hybridized DNA fragment.

Using the amino acid sequences 1 to 3 as a sense primer and an antisense primer as described above, the nucleotide sequence encoding the amino acid sequence sandwiched by these primers is propagated as a DNA fragment for a plurality of bacteria. The nucleotide sequence is determined by a known method such as the dideoxy method.

Such nucleotide sequences differ between homologous species and between strains, and in contrast to the aforementioned 16S rRNA nucleotide sequences that differ only slightly between homologous species and between strains, comparisons between the same species were made. In many cases, there is a remarkable difference. Therefore, bacteria can be identified by using such a DNA fragment as a signature base sequence.

Since the base sequence amplified by PCR has the above specificity as described above, the base sequence obtained as described above may be used as a probe as it is. Furthermore, when a base sequence present in a closely related bacterial species is obtained in the same manner and a multiple comparison is carried out, a very highly specific probe used for identification at the species or strain level can be obtained. Using these probes, the target bacteria are specifically detected and identified from the natural flora containing many unknown bacteria. In addition, these probes can also be used to screen for unidentified new species.

The bacteria that can be detected and identified by the method for detecting and identifying microorganisms of the present invention are not particularly limited as long as they have dioxygenase. However, an aromatic-decomposing bacterium that degrades an aromatic compound is preferred. Aromatic degradation bacteria are biphenyl, toluene,
Alternatively, it refers to a bacterium that requires an aromatic compound such as naphthalene as a substrate and decomposes it. Specifically, Pseudomonas genus, Alcaligens (Alcaligen)
es), Rhodococcus, Acinetobacter, Sphingomonas and the like. The present invention can be suitably used for detection and identification of such aromatic degrading bacteria.

Further, biphenyl, toluene, naphthalene and the like are aromatic compounds contained in the crude oil, and when the crude oil is contaminated as described above, the amounts of these compounds in the crude oil can be measured simply and quickly. Difficult to do. Therefore, by examining the growth of aromatic compounds in crude oil that degrades these compounds by the detection method and the identification method of the present invention, it is possible to monitor how much the biological decomposition of crude oil has progressed.

[0024]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Determination of Amino Acid Sequence Having Conservation The amino acid sequences of 11 types of aromatic oxygen-degrading bacteria dioxygenase genes were searched with SWISS PROT, and alignment was performed using Gene Works software to confirm the conservation. The presence of the amino acid sequence was examined. It was revealed that there are a plurality of conservative amino acid sequences on the large subunit of the amino acid sequence of dioxygenase, and these amino acid sequences were found to be composed of 6 amino acids and 7 amino acids, respectively.

As a result of comparing the above-mentioned amino acid sequences, the amino acid sequence consisting of 6 amino acids was designated as amino acid sequences 1 and 2 and the amino acid sequence 3 consisting of 7 amino acids located downstream thereof, and the results are shown in FIGS. Was.

By determining and comparing the amino acid sequences as described above, the first and fourth to sixth to sixth amino acids from the N-terminal side of these amino acid sequences in amino acid sequences 1 and 2 are determined.
The third underlined amino acid sequence is a highly conserved region, and in the amino acid sequence 3, the first to third and seventh underlined amino acid sequences from the N-terminal side of this sequence are conserved. Is a high region.

Pseudomonas putida F1 strain (SWISS PR
The amino acid sequence of the gene of the large subunit of 1,2-dioxygenase (OT) was determined by the method described above and compared. This sequence was found to correspond to amino acids 116-121 and 208-214 of the amino acid sequence of the 1,2-dioxygenase gene of the above strain.

Example 2 Design and Preparation of Primers A nucleotide sequence corresponding to the amino acid sequence shown in FIGS. 1 and 2 was determined, and based on this, a sense consisting of the nucleotide sequence shown in the frame of FIGS. 1 and 2 was prepared. Primers and antisense primers were designed.

The designed sense primer contains a nucleotide sequence encoding the above conserved amino acid sequence,
The same applies to the antisense primer. But,
In the antisense primer, although it is important that the leading edge of the primer is specific, the third base of the codon corresponding to glutamic acid corresponding to the C-terminus of the primer is non-specific. A sequence from which the third base was deleted was used.

As described above, the 18mer used for PCR
Were prepared as a mixture of 128 types. These primers are shown in FIG. In FIG. 3, bases in parentheses indicate which bases can be used when there are a plurality of codons encoding the amino acids shown in FIGS.

Example 3 Detection and Identification of Aromatic Degrading Bacteria Using Primers Using the sense primers and antisense primers prepared in Example 2, PCR was performed using the DNA of the following aromatic degrading bacteria as a template. went.

(1) DN of microorganism used as template
A: (a) DNA of biphenyl-degrading bacteria; Pseudomonas sp.
strain ATCC53643, P. pseudoalkaligenes KF707, Alcaligenes sp. strain ATCC53640 (b) DNA of a naphthalene-degrading bacterium; Pseudomonas sp.
strain ATCC17483, Pseudomonas putida ATCC1748
4. Pseudomonas putida PpG7 (c) DNA of toluene-degrading bacterium; Pseudomonas putida mt-2 (d) DNA of benzene-phenanthrene-degrading bacterium; MBI
1340, MBI 1422, NAD1 (f) DNA of phenanthrene-degrading bacterium; Sphingomonas sp. Strain TB4

Among the above-mentioned bacteria, two strains of Pseudomonas putida mt-2 and Pseudomonas putida PpG7 are 1,2-
This strain has a dioxygenase gene on a plasmid. On the other hand, Pseudomonas sp. Strain ATCC53643, Pseudomonas sp. Strain ATCC17483, Pseudomonas putida KT2440, Pseudomonas putida ATCC17484, Pseudomonas pseudoalkagenes KF707, Alcaligenes sp. Strain ATCC53640, Pseudomonas putida
PB4, Sphingomonas sp. Strain TB4, unidentified hydrocarbon-degrading bacteria (MBI 1340, MBI 1422, and NAD1)
One strain has a dioxygenase gene on the chromosome.

(2) Culture of bacteria Sphingomonas sp. Strain TB4, Pseudomonas sp.
Putida PB4, an unidentified hydrocarbon-degrading bacterium (MBI 1340, MBI
For the culture of 1422), Marine broth (Marine broth (Difco) containing 37.4 g, sodium chloride 30.0 g and distilled water 1000.0 ml) was used.

Pseudomonas Pseudoalkaligenes
KF707, Pseudomonas putida PpG7, Pseudomonas s
p.strain ATCC53643, Alcaligenes sp.strain ATCC
For culture of 53640, Pseudomonas putida mt-2 and Pseudomonas putida KT2440, tryptic soy broth (30.0 g of tryptic soy broth (Difco) and 1000.0 ml of distilled water)
Was used.

For the culture of Pseudomonas sp. Strain ATCC17483 and Pseudomonas putida ATCC17484, a nutrient medium (meat extract 10.0 g, peptone 10.0 g,
1.0 g of sodium chloride and 400.0 ml of distilled water). Each strain was cultured with shaking at 28 ° C. until the stationary phase was reached.

(3) Isolation of the dioxygenase gene from the bacterium The dioxygenase gene was isolated from the above bacterium by the following procedure. Each of the above bacteria was cultured as described above, and 6 ml of the medium was taken and centrifuged at 6,000 rpm for 10 minutes to remove the supernatant. 120 μl of 10% SDS, 24 μl of proteinase K (10 m
g / ml), resuspend the precipitate in 2 ml of TE buffer containing
Incubated for 1 hour at ° C. 5M sodium chloride 40
0 μl was added and mixed. Then, 320 μl of CTAB / NaCl solution
Was added and incubated at 65 ° C. for 20 minutes.

The mixture was extracted with an equal volume of chloroform / isoamyl alcohol and centrifuged at room temperature for 10 minutes at 7,000 rpm. Then, extract with an equal volume of TE saturated phenol, at room temperature for 10 minutes,
Centrifuged at 7,000 rpm. 0.6 volume of isopropanol for DN
A was precipitated and washed with 70% ethanol. The supernatant was removed and the precipitate was resuspended in 4 ml of TE buffer. 20 μl R
Nase (20 mg / ml) was added and incubated at 37 ° C. for 30 minutes. Then, the mixture was extracted with an equal volume of TE-saturated phenol and centrifuged at 7,000 rpm for 10 minutes at room temperature.

The mixture was extracted with an equal volume of chloroform / isoamyl alcohol, and centrifuged at room temperature for 10 minutes at 7,000 rpm. Then, extract with an equal amount of TE-saturated phenol, and at room temperature for 10 minutes,
Centrifuged at 7,000 rpm. 0.6 volume of isopropanol for DN
A was precipitated and washed with 70% ethanol. The supernatant was removed and the precipitate was resuspended in 4 ml of TE buffer to obtain the DNA of the above-mentioned bacteria.

(4) Isolation of plasmid DNA TNS buffer (containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl and 20% sucrose), lysis buffer (10
0 mM Tris-HCl (pH 8.0), 50 mM EDTA, 0.5 M NaCl, and 2% SDS), RNase buffer (0.3 mM pancreatic RNase A
M EDTA containing 0.1M sodium acetate (pH 4.8) solution
mg / ml and heated to 80 ° C for 10 minutes),
3M NaOH and 2M Tris-HCl (pH 5.0) were prepared, and plasmid DNA was isolated using these reagents according to the following procedure.

Take 500 ml of the culture supernatant of each of the above bacteria, and
The mixture was centrifuged at 4 ° C. for 20 minutes using rpm (Sorval, GSA rotor). The pellet was frozen at −20 ° C. and thawed at room temperature. The pellet was resuspended in 100 ml of TNS buffer, 40 ml of lysozyme (dissolved at 10 mg / ml in TE buffer) and 3 ml of RNase buffer were added and incubated on ice for 30 minutes. Then, 120 ml of lysis buffer was added, incubated well at 25 ° C. until the solution became clear. 3M NaOH was added dropwise and the pH was adjusted to 12.1 with gentle stirring, and then 2M Tris-HCl
(PH 5.0) was added slowly to adjust the pH to 8.5. The volume of the solution was measured and 5.8 g of solid NaCl per 100 ml of solution was added to a final salt concentration of 1 M and incubated on ice for 1 hour.

The mixture was centrifuged at 8,000 rpm (Sorval, GSA rotor) at 4 ° C. for 20 minutes, and the volume of the obtained supernatant was measured.
Add PEG6,000 to a final concentration of 5% (w / v) and add 4 ° C
At rest overnight. The obtained precipitate was subjected to 10,000 rpm (Sorval,
The mixture was centrifuged at 4 ° C. for 30 minutes using a GSA rotor, and the supernatant was decanted off. The pellet was resuspended in 10 ml of TE buffer, transferred to another tube and extracted three times with an equal volume of phenol / chloroform. The resulting aqueous layer was washed with chloroform /
Extracted three times with n-amyl alcohol. The obtained aqueous layers were combined, CsCl and ethidium bromide were added, and the mixture was appropriately adjusted according to the plasmid whose density was to be obtained.

The mixture was centrifuged at 50,000 rpm (Sorval, TV-865 rotor) at 20 ° C. for 15 hours to obtain a supercoiled plasmid DNA from contaminants. Ultraviolet rays were applied to the centrifuge tube, DNA was taken out with a syringe, and this DNA was subjected to density gradient centrifugation as described above to obtain a purified plasmid DNA. Ethidium bromide was removed by water-saturated n-butanol extraction, and CsCl was removed by dialysis against 4 L of TE buffer for 24 hours.

Using the plasmid DNA obtained as described above as a template, specific DNA fragments were grown by PCR to detect these bacteria.

The PCR conditions are as follows. (1) Reagents used 25 μl of 10 × PCR buffer (100 mM Tris-HCl (pH 8.
3), containing 500 mM KCl, 15 mM MgCl ₂ , 0.01% gelatin (w / v)) 0.5 μl sense primer (100 pmol) 0.5 μl antisense primer (100 pmol) 2.5 μl dTNPs (2 mM) 1 unit Taq Polymerase 250 ng of template DNA Sterile water was added to the above reagent to make the total volume of the solution 25 μl, and PCR was performed under the following reaction conditions.

(2) Reaction conditions 45 cycles were carried out at 94 ° C. for 1 minute, 94 ° C. for 30 seconds, 55 ° C. for 1 minute and 72 ° C. for 1 minute, followed by 2 cycles at 72 ° C. for 2 minutes.

Using the DNA of these bacteria as a template, P
All of the amplification products obtained as a result of CR had approximately the same length of about 318 bp. From the above, it has been clarified that any bacteria having the dioxygenase gene on the chromosome or on the plasmid can be detected using the conserved amino acid sequence.

Further, a bacterium having the above substrate specificity is
In order to use biphenyl and naphthalene as sole carbon sources, an M9 medium excluding glucose was prepared, and a carbon source was added. By the plate method, each of the above bacteria is cultured at 28 ° C,
The growth ability of these bacteria when biphenyl and naphthalene were used as carbon sources was examined. At this time, the gene of each bacterium was amplified by PCR, and the nucleotide sequences of the amplified products were compared using the identification method of the present invention. Table 1 shows the results. These results revealed that the substrate specificity was consistent with the length of the gene amplification product, and that this was used to identify and discriminate bacteria.

[0050]

[Table 1]

[0051]

According to the method for detecting a microorganism of the present invention, a microorganism having a dioxygenase gene can be easily and quickly detected. In addition, the microorganism identifying method of the present invention can easily, quickly, and accurately identify a bacterium that decomposes a target aromatic compound without using a conventional biochemical test.

Further, according to the monitoring method of the present invention, aromatic decomposing bacteria can be specifically detected from soil or the like contaminated with crude oil. Therefore, it is possible to accurately detect how much the aromatic-degrading bacteria in such a sample is growing, and it is possible to use the growth of such aromatic-degrading bacteria as an index of the crude oil decomposition process. By appropriately selecting a probe used in the method for detecting a microorganism of the present invention, a target microorganism can be reliably detected from a flora containing a large number of microorganisms. Therefore, it can also be used for diagnosis of infectious diseases, etc., in which it is necessary to track the fate of specific bacteria.

As described above, the present invention is not only applied to the biomeridation technology for crude oil-contaminated soil as described above, but also for the development of an environmental purification system using microorganisms, the management of food production processes in the food industry, and the like. It is also possible to apply to.

[Brief description of the drawings]

FIG. 1 is a diagram showing a conserved amino acid sequence of 1,2-dioxygenase.

FIG. 2 is a diagram showing another conserved amino acid sequence of 1,2-dioxygenase.

FIG. 3 is a diagram showing sense primers and antisense primers used for PCR.

Claims (7)

(57) [Claims] 1. A aromatic ring hydroxylase dioxygenase A
A nucleotide sequence coding for a region in the amino acid sequence that is conserved among the microorganisms to be detected, or its complement
A method for detecting a microorganism using a simple base sequence . 2. The method according to claim 1, wherein the microorganisms to be detected are preserved.
There are two regions having the same amino acid sequence, and one of the amino acid sequences of these two regions is Cys-Ser-Tyr-His-Gl.
y-Trp or Cys-Ser-Phe-His-G
an amino acid sequence consisting ly-Trp, the other As
n-Trp-Lys-Phe-Ala-Ala-Glu
An amino acid sequence represented by the formula:
The method for detecting a microorganism according to claim 1 . 3. An aromatic ring hydroxylase dioxygenase a.
Conserved among the microorganisms to be detected in the amino acid sequence
Base sequence encoding two regions having
After detecting the microorganism using the complementary base sequence,
A method for identifying a microorganism using a base sequence encoding an amino acid sequence sandwiched between two regions . 4. One of the amino acid sequences of the two regions is an amino acid sequence consisting of Cys-Ser-Tyr-His-Gly-Trp or Cys-Ser-Phe-His-Gly-Trp, and the other is Asn-Trp. -L
The method according to claim 3, wherein the amino acid sequence is represented by ys-Phe-Ala-Ala-Glu . 5. The method according to claim 3, wherein the microorganism is an aromatic degrading bacterium. 6. The aromatic ring hydroxylase dioxygenase A
A nucleotide sequence coding for a region in the amino acid sequence that is conserved among the microorganisms to be detected, or its complement
With a base sequence, partial aromatic compounds contained in the crude oil
A method of detecting the growth of aromatic degrading bacteria to be decomposed and monitoring the biological decomposition of crude oil using the detected amount as an index. 7. The method according to claim 7, wherein the microorganisms to be detected are conserved.
There are two regions having the same amino acid sequence, and one of the amino acid sequences of these two regions is Cys-Ser-Tyr-His-Gl.
y-Trp or Cys-Ser-Phe-His-G
an amino acid sequence consisting ly-Trp, the other As
n-Trp-Lys-Phe-Ala-Ala-Glu
The method according to claim 6, wherein the amino acid sequence is represented by the following formula: