JP2692015B2 - Genes that enhance transformation using mercury-resistant shuttle vector plasmids for Thiobacillus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to gene manipulation of Thiobacillus, and more specifically, in a method of using mercury resistance as a selection marker, a DNA fragment of a regulatory gene, a recombinant plasmid, and this plasmid which greatly enhance it. And a host cell transformed with this shuttle vector plasmid.

[0002]

2. Description of the Related Art Conventionally, Thiobacillus
Genus fungi are often found near sulfide deposits and are used for bacterial leaching of ores. Thiobacillus is ferrous (Fe ²⁺ )
Since it has the ability to oxidize iron to ferric iron (Fe ³⁺ ), this oxidizing ability can be used to treat mine wastewater, deironization in hydrometallurgy or treatment of reducing gas such as H ₂ S gas. It has been used and has been used as a very useful bacterium industrially.

In the above-mentioned applications, Thiobacillus ferrooxidans (Thiobacillus f), which is a bacterium belonging to the genus Thiobacillus, is an autotrophic bacterium.
The usefulness of errooxidans) is recognized. However, the growth rate of T. ferrooxidans is
It is extremely slow compared with aerobic bacteria such as erotrophic bacteria. For example, when compared with E. coli, the doubling time is about 30
The cell yield in the quantitative medium is about 1/60. for that reason,
When this bacterium is used for bacteria leaching, wastewater treatment, etc., a large field and container are required, resulting in an increase in manufacturing cost and treatment cost, and the use of this bacterium has been severely restricted.

Therefore, similar to the case of using other microorganisms, random screening was performed to search for bacteria having a fast growth rate, but no great result was obtained. On the other hand, attempts to construct new strains have been made all over the world by genetic engineering that requires a vector having an appropriate selection marker to select useful bacteria, and the following are already known. Rawlings, etc., construction of a new strain using the chloramphenicol resistance gene of other fungi, construction of a new strain using the arsenic resistance gene of other fungi by the same person, and Pseudomonas spp. There is a construction of a new strain using the mercury resistance of the bacterium, etc., and for details of these, see Japanese Patent Laid-Open No.
0-91988, JP-A-60-102189 and JP-A-62-44773.

However, in all of these, the gene serving as a selection marker of the vector is not a gene of a Thiobacillus bacterium, but a gene of another bacterium, so that the expression efficiency of the thiobacillus bacterium in a cell is poor. was there.

[0006] Therefore, as a remedy for the above problems, the present inventors cloned the mercury reductase gene present on the genomic DNA of T. ferrooxidans E-15 strain of the genus Thiobacillus as a selection marker. 7, Figure 8
And pTMB631, pTMB632, pTMA641 and
A shuttle cloning vector such as pTMA642 was created. For details, see Japanese Patent Laid-Open No. 2-167082.
Is disclosed.

Furthermore, thereafter, the mercury resistance gene (mer ^r ) fragment of the T. ferrooxidans E-15 strain was present in the merA, which is the mercury reductase gene, and in the intracellular cytoplasm upstream of this merA. It was revealed by the present inventors that it is composed of a gene called merC that makes a protein that controls the transport of mercury between cells.

[0008] The above-mentioned improvement measures by the present inventors have used a homogeneous gene of Thiobacillus as a selection marker in the production of a vector plasmid containing a mercury resistance gene. Therefore, a vector produced by using a heterogeneous gene as a selection marker. The drawbacks of the plasmid could be eliminated. That is, the efficiency of mercury resistance expression in the cells of Escherichia coli and Thiobacillus cells was improved.

However, the development of the mercury resistance of the above improvement measures was not sharp as expected, and specifically, there were the following defects. Since the expression of mercury resistance is affected by the physiological state of the host cell, it is difficult to adjust the conditions for transformation, and the frequency of finding transformants is low. Also,
Depending on how the mercury load is applied, an unstable phenomenon such as death of host cells, loss of plasmid, or transfer of mercury resistance marker to genomic DNA may occur.

[0010]

DISCLOSURE OF THE INVENTION The present invention solves the problems of the above-mentioned conventional techniques, transforms a cloning vector having a mercury-resistant selection marker for T. ferrooxidans at a higher frequency, and It is an object to provide a DNA element and a host cell which can be stabilized in the state of a vector plasmid.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the problems of the above-mentioned conventional techniques, and found that a regulatory gene for a mercury-resistant operon in Thiobacillus ferrooxidans is present. The present invention could be provided by finding, cloning and inserting it into a vector plasmid.

The details of the present invention are shown below.

First, in order to determine the structure of the mercury-resistant operon in T. ferrooxidans, T. ferrooxidans E-15 was prepared by Northern blotting.
M of the mercury resistance gene (mer ^r ) in the genomic DNA of the strain
RNA size was analyzed. As a result, the transcript of the mercury resistance gene has a size of about 2.5 kb, which is encoded by a gene for a protein (merC) that controls mercury transport between the outside world and cells and a mercury reductase gene (merA). It was slightly larger. Therefore, merC and merA
Was probably transcribed at the same time. However, transcription was performed in cells that were induced by mercury, but was hardly performed in cells that were not induced.

On the other hand, when E. coli is transformed with a recombinant vector consisting of the merC-merA gene of a plasmid (Japanese Patent Laid-Open No. 2-167082) as shown in FIG. It was expressed constitutively even without induction.

That is, as described above, T. ferrooxidans
In T. ferrooxidans, the fact that the expression of mercury resistance (mer ^r ) is regulated at the transcriptional level in
It means that there is a gene that does some kind of regulation.

Generally, a bacterial mercury resistance gene (mer ^r ) is composed of a system containing a mercury resistance enzyme gene (merA), and a regulatory gene merR is followed by an operator and a promoter sequence, and further merT, merP and merA. And me
The gene called rD is lined up. In the R100 mer ^r system, a gene called merC exists between merP and merA. As described above, the mercury resistance genes found so far generally have an operon structure. However, in T. ferrooxidans, it was not possible to detect the presence of merR, which is an expression-regulating gene, near the DNA fragment in which merC and merA were present.

Therefore, colony hybridization (colony hybridiza) is performed using a known merR gene as a probe.
in the genomic DNA of T. ferrooxidans.
The rR gene was searched.

First, the T. ferrooxidans E-15 strain was subjected to colony hybridization using the merR gene of the genus Pseudomonas as a probe. However, only a very weak signal could be obtained and the merR gene could not be selected. It was speculated that this was because the E-15 strain had low homology with the merR of the genus Pseudomonas as a probe.

Next, when the above-mentioned method was applied to the T. ferrooxidans E-6 strain, it showed high homology.
The merR gene could be selected from the BamHI library of idans genomic DNA.

The selected T. ferrooxidans mercury resistance regulatory gene (merR) fragment finally contains linkers (EcoRI, BamH).
I, SalI) was added, and a fragment containing the merR gene of about 0.6 kb was cloned into pUC18 of E. coli plasmid to prepare a new plasmid (pTMR3B (Fig. 1), pTMR3E).
(Fig. 2), named pTMR3S (Fig. 3)).

T. ferroox which could not select the merR gene by the above colony hybridization
For the idans E-15 strain, the merR gene was searched by the following method.

First, pT was added to the BamHI site of pAYCYC184, which is a plasmid incompatible with pUC18 of E. coli plasmid.
A BamHI fragment containing the merC-merA gene of T. ferrooxidans contained in M314 was inserted to prepare a chimeric plasmid pAT116. Next, Escherichia coli DH5α strain was transformed with the prepared pAT116, and this was treated by a method such as Hanahan to obtain competent cells.

On the other hand, T. fer cut with EcoRI and PstI
The genomic DNA of rooxidans E-15 strain was ligated to the DNA of pUC18 to prepare EcoRI and PstI libraries.
Both of the prepared libraries were cloned into the above competent cells, and a clone having an increased mercury resistance was selected. As a result, a clone containing an EcoRI fragment of about 1.0 kb and a PstI fragment of about 1.7 kb was obtained. HgCl of both obtained clones
_When the MIC (minimum inhibitory concentration) for ₂ was measured,
It was 20 μg / ml or more. This is pAT116 MIC 5 ~
7.5 μg / ml (for those that do not contain the merC-merA gene at all
2.5 to 5 μg / ml), clearly merC
-It was shown that a factor that enhances the merA gene, that is, a mercury resistance regulatory gene merR was included.

The new plasmid containing the PstI fragment obtained above was named pTMR15 (FIG. 4), and the new plasmid containing the EcoRI fragment was named pTMR25 (FIG. 5).

In addition, pTMR3B prepared by the above method,
The inserts of pTMR3E, pTMR3S, pTMR15 and pTMR25 containing the mercury resistance gene were DNA sequenced and the merR gene part of these plasmids was determined by comparison with the known merR gene.

As a result, it was revealed that the merR gene was encoded in the portions shown in FIGS. 31, 32 and 33. FIG. 31 shows merR genes of pTMR3B, pTMR3E and pTMR3S (referred to as merR (1) to distinguish them from others), and FIG. 32 shows pTMR15.
MerR gene (named merR (2) to distinguish it from others),
And Figure 33 shows the merR gene of pTMR25 (me
rR (3)). Note that FIG. 31, FIG. 32, and FIG.
R is EcoRI, Sm is SmaI, 47 is EcoR47 III, H
Represents Hind III, Sp represents SphI, K represents KpnI, Xb represents XbaI and V represents EcoRV.

Furthermore, the nucleotide sequence of the DNA fragment encoding the mercury resistance regulatory gene of Thiobacillus, that is, FIG.
MerR coded at locations shown in Figure 31, Figure 32, and Figure 33
The gene portion and its translated amino acid are shown in Chemical formula 7, Chemical formula 8 and Chemical formula 9. The upper row of these sequences is the base sequence, and the lower row is the amino acid sequence.

The merR gene portion shown in FIG. 31 is the merR
(1), the merR gene part shown in FIG. 32 is merR (2), and the merR gene part shown in FIG. 33 is merR (3).

[0029]

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[0030]

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[0031]

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MerR (1) has a high homology of about 92% with the merR gene of Tn501.
(2) and merR (3) are about 57 with the merR gene of Tn501.
The homology was as low as%. Also, with merR (2)
There were only three differences in the amino acid sequence from merR (3), which were very similar.

Next, the inventors of the present invention described the above merR (1), me.
FIG. 6 shows rR (2) or merR (3) pTMB631, FIG.
PTMB632 shown in Fig. 8, pTMA641 shown in Fig. 8, pTM shown in Fig. 9
A642 (these are known vectors described in JP-A No. 2-167082) or pTMI38 shown in FIG. 18 (pKT2 which is a wide host range vector of the incompatibility group Q system as an origin of replication).
40 merC-merA gene-ligated vector) and the replication origin of the Thiobacillus-derived plasmid and the replication origin of the Escherichia coli-derived plasmid or the replication origin of the broad host range vector belonging to the incompatibility group Q. A new shuttle vector plasmid containing a regulatory gene, a gene of a protein that controls mercury transport between the outside world and the inside of the cell, and a group of mercury resistance genes of the genus Thiobacillus consisting of a mercury reductase gene was prepared. In addition, pTMI38 in the above sentence
And pKT240 (well-known commercially available product) are cleaved with restriction enzymes EcoRI (well-known commercially available product) and SphI (well-known commercially available product), and the resulting longer DNA fragment (12.4 kb) is recovered. PTM315 (described in Japanese Patent Application Laid-Open No. 2-167082)
Known vector) of which the EcoRI-SphI fragment of 4.6 kb (both cleavage planes are derived from the multiple cloning site of pVC18 (well-known commercial product)) was ligated, and this hybrid was ligated to pTMI38 (17.0 kb). It was named.

MerR (1), merR (2) and merR
Although the insertion position of (3) varies depending on each vector, merC
-Do not cut the merA gene in the middle, and
It is a part that is not involved in the origin of replication of rooxidans. The new shuttle vector plasmid prepared, the type of the introduced merR gene and its introduction site are shown in the table below.

In the production of such a novel shuttle vector plasmid, when inserting the merR gene, T.
As long as the conditions do not affect the ferrooxidans and E. coli plasmid replication regions, and do not affect the T. ferrooxidans-derived merC-merA gene, there is a large difference in the basic functions of the shuttle vector plasmids produced even if the insertion positions differ. Does not occur.

[0036]

The mercury resistance of the vector containing the merR gene of the plasmid of the present invention was compared with the mercury resistance of the vector containing only the merC-merA gene of the conventional plasmid. In the comparison, as a plasmid of the present invention, T. ferric
pTSB121 and pTNA as origin of replication for rooxidans
33 or pKT240, which has a merC-merA system, was shown as a means to solve the problem merR (1)
Alternatively, merR (2) was used in place.

First, Escherichia coli DH containing the vector of the present invention
The 5α strain was used as a L. B. It was solidified with a petri dish. Then filter paper containing HgCl ₂ different concentrations thereon (Whatman A.A. disk 6.
(0 mmφ) and place it overnight at 37 ° C. Measure the diameter of the inhibition circle (clear part where cells could not grow) and subtract the original disc diameter (6.0 mm). The results were plotted and the results are shown in Table 1.

[0038]

[Table 1]

As can be seen from this table, the plasmid of the present invention containing the merR gene has a much smaller region of growth inhibition than the conventional plasmid containing only the merC-merA gene, and the resistance to mercury is significantly increased. It was confirmed that it was enhanced.

Furthermore, a time factor was added to the above test to compare the enzyme activities of both vectors.

First, Escherichia coli DH containing the vector of the present invention
The 5α strain was cultured to a logarithmic growth phase in a medium without mercury, 10 μg / ml of mercuric chloride was added at a predetermined time point, and the activity of the mercury reductase per unit protein after that was plotted. The results are shown in the table below.

As can be seen from this table, the conventional merC-me
It was confirmed that the vector containing the merR gene of the present invention had a higher enzymatic activity in a shorter time than the vector containing only the rA gene.

Next, the transformation efficiencies of the vector containing the merR gene of the present invention and the conventional vector containing no merR gene were compared based on the electroporation method. First, as a host cell for transformation, T. f in the logarithmic growth phase is used.
Both vectors were introduced into the iron-oxidizing bacteria collected from the Y4-3 strain of errooxidans and cultured on a silica gel plate containing 0.25 μg / ml of HgCl ₂ . Next, the colonies that appeared on this plate were separated, and transformants with this vector were selected. The results are shown in the table below.

As can be seen from this table, the number of the transformants of the vector containing the merR gene of the present invention was merC-me.
It was about 50 times that of the conventional vector containing only the rA gene, and it was confirmed that the transformation efficiency was remarkably improved. In addition,
The transformation efficiency when pTMZ32 or pTMZ36 is introduced is
It was about 4.0 × 10 ² cells / μg Vector DNA.

Further, the colonies on the silica gel plate were transferred to a liquid medium containing a slight amount of mercury and allowed to grow, and the transfer rate from the colonies to the liquid medium was calculated. As a result, the transfer rate of the vector containing only the merC-merA gene was 50%.
The stability was low as follows, and it was shown that the transfer rate of the vector to which the merR gene of the present invention was added was as high as 100%.

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

[0047]

Example 1 Recovery of genomic DNA from T. ferrooxidans in the present invention will be described.

First, T. ferrooxidans E-6 strain and E-15
Each of the strains was inoculated into 250 ml of 9K medium of Silverman et al. And cultured at 30 ° C. for 2 days. After culturing, cells were collected by centrifugation, low pH cleaning solution; 2 times (9K mineral salts medium containing MgSO ₄ · 7H ₂ O in 0.16M adjusted to pH 1.9 with sulfuric acid), high pH
Washing solution (25 mM phosphate buffer containing 0.3 M sucrose, 10 mM EDTA; pH 8.0) once, and then 50 mM phosphate buffer (pH 7.
It was washed once in 4).

Next, 4 ml of a solution consisting of 2.5 mg / ml lysozyme, 0.05 M glucose, 25 mM Tris-HCl (pH 8.0) and 10 mM EDTA was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. 5 more
Add 00 μl of 0.25M EDTA, leave at 0 ° C for 10 minutes, then 500
Lysis was performed by adding μl of 10% SDS. 50 μl of 20
Add 37 mg / ml proteinase K
The protein was decomposed by incubating at 0 ° C. for 2 hours.
Add an equal volume of phenol: chloroform (1: 1) to the solution.
After the mixture was added and deproteinization was performed three times, the nucleic acid portion was precipitated with isopropyl alcohol, collected by centrifugation, and dried. Furthermore, in order to purify it,
Density gradient centrifugation using cesium chloride (20 ° C, 55000 rpm,
16 hours) and dialysis (day and night), purified genomic DN
A was obtained.

[0050]

Example 2 T. ferrooxidan recovered by the method of Example 1.
The cloning of the merR gene from s E-6 strain genomic DNA is described below.

First, 1 μg of E-6 strain genomic DNA and 0.2 μg of E. coli plasmid pUC18 DNA were digested with the restriction enzyme Ec.
was cut with EcoRI, these fragments were ligated using T _4-DNA ligase. This ligated DNA mixture was used for E. coli DH
Incorporated into 5α cells.

On the other hand, ampicillin 50 μg / ml, X-gal 40
Plate of LB medium containing μg / ml and isopropyl-β-D-thiogalactopyranoside 1 mM (1.5% agar)
A nylon membrane filter (BNNG82, Amersham Chemicals) of the same type as the petri dish was placed.

Escherichia coli DH5α cells incorporating the above-mentioned ligated DNA mixture were coated on the prepared medium and cultured at 37 ° C. for a whole day and night. After culturing, the known colony merR gene of Tn501 was used as a probe for the obtained colonies, and the method of Granstein and Hognes (M. Granstein and DS Hogn
ess, Proc. Natl. Acad. Sci. USA 77: 3961-3965,197
Colony hybridization according to 5),
About 6.5 kb of T. ferrooxidans genome D from a positive colony
PTMR21 containing NA was obtained.

The merR gene portion of the obtained pTMR21 was DN
Southern hybridization (PS Thomas, Proc. Natl. Acad.
Sci. USA 77 : 5201-5205, 1980), etc.
It was confirmed to be a region of about 0.6 kb sandwiching the site.
So we cut out this fragment and used BamHI, EcoRI or Sa
Each linker of lI was added, and each was cloned into E. coli plasmid pUC 18 to obtain a new shuttle vector plasmid. The shuttle vector plasmids obtained were pTMR3B (Fig. 1), pTMR3E (Fig. 2) and pTMR3S.
(Fig. 3).

[0055]

[Example 3] T. ferrooxida recovered by the method of Example 1
The cloning of the merR gene from the ns E-15 strain genomic DNA is described below.

According to the method described in Example 2, cloning of the merR gene from E-15 strain genomic DNA gives a weak signal in colony hybridization, and the desired product cannot be obtained. Therefore, merC-merA
A selection method was used depending on whether the strain carrying the gene increases mercury resistance.

First, of T. ferrooxidans contained in the BamHI fragment of pACYC184 which is incompatible with E. coli plasmid pUC18
A fragment containing the merC-merA gene was ligated together to prepare a chimeric plasmid pAT116. Next, Escherichia coli DH5α strain was transformed with this chimeric plasmid pAT116. The cells obtained by the transformation were treated by a method such as Hanahan to prepare competent cells.

On the other hand, genome D of T. ferrooxidans E-15 strain
NA 1 μg and E. coli plasmid pUC18 DNA 0.2
μg was cleaved with restriction enzymes EcoRI and PstI, and these fragments were ligated with T ₄ -DNA ligase. This EcoR
The ligated DNA mixture containing the I fragment and the ligated DNA mixture containing the PstI fragment were cloned into competent cells of E. coli DH5α strain containing pAT116 described above. This was plated with LB medium containing 10 μg / ml HgCl ₂ (1.5%
Agar) and cultured overnight at 37 ° C.

After culturing, the obtained colonies were analyzed, and it was confirmed that two independent clones containing an EcoRI fragment of about 1.0 kb and a PstI fragment of 1.7 kb were obtained. The bacterial cells obtained by the above transformation are 5 to 7.
Both clones showed resistance of 20 μg / ml HgCl ₂ or more, while resistance of 5 μg / ml HgCl ₂ showed.
-It was confirmed that a merR gene that enhances the merA gene was included.

The shuttle vector plasmid containing the PstI fragment thus prepared was pTMR15 (FIG. 4), and the shuttle vector plasmid containing the EcoRI fragment was pTMR15.
It was named R25 (Fig. 5).

[0061]

Example 4 In this example, the nucleotide sequence and amino acid sequence of the DNA encoding the mercury resistance regulatory gene merR of T. ferrooxidans will be described.

The nucleotide sequence was determined by the dideoxynucleotide chain-termination method (Sanger method) using a denatured plasmid template (Hattori and Sakaki, 1986). Instead of dGTP, dc ⁷ dGTP
Including 7-deaza sequencing kit (Takara Shuzo) and (α
- ³² P) dCTP (NEW or Amersham chemicals: 4000Ci / mmol)
It was used. In addition, 16 bp upstream of Hind III of pUC18 and EcoR
Primer M1 hybridizing with 8 bp upstream of I
(15mer, 5'-AGTCACGACGTTGTA-3 ') and primer RV
(17mer. 5′-CAGGAAACAGCTATGAC-3 ′) was obtained from Takara Shuzo.

On the other hand, the amino acid sequence of SDC-GENETYX
genetic information processing program (Software d
evelopment).

[0064] T. ferrooxid determined by the above method
The nucleotide sequence and amino acid sequence of the DNA fragment encoding the ans merR gene are as described in claim 1 of the claims.

[0065]

[Example 5] A gene having a replication origin of a Thiobacillus-derived plasmid and an Escherichia coli-derived plasmid of the present invention and controlling the transport of mercury between the outside world and cells and a mercury reductase gene. An example of a method for producing a shuttle vector plasmid containing the thiobacillus mercury resistance gene group consisting of is described below.

First, pTMB6 having BamHI sites at two sites
31 (FIG. 6) was cut by partial cutting (2 μg). Since the cleaved DNA has one of two BamHI sites cleaved, it was separated by agarose gel electrophoresis, and only the fragment showing 11.3 kb was excised and recovered.
About 0.5 μg of the desired fragment was obtained.

The obtained fragment was 1 mM dXTP and the enzyme Kleno.
The BamHI site was blunt-ended in the presence of w fragment, ligated with T ₄ -DNA ligase, and transformed into Escherichia coli DH5α strain. The cells obtained by the transformation were applied to a solid medium containing ampicillin 50 μg / ml and cultured.

After culturing, the obtained colonies were cultivated again in a liquid medium to extract DNA. The extracted DNA is BamH
It was cleaved with I and selected one that retained one of the two BamHI sites that were present in pTMB631 and selected pTMB633 (Figure 10) and p
TMB634 (Figure 11) was obtained.

On the other hand, pTMR3B (3 μg) containing the merR gene of T. ferrooxidans E-6 strain as an EcoRI fragment was added to BamHI.
Cut. The cleaved DNA was separated by agarose gel electrophoresis in the same manner as above, a BamHI fragment of about 0.6 kb was excised and recovered, and a target fragment containing about 0.5 μg of the merR gene was prepared.

PTMB633 thus obtained (FIG. 10)
Both the DNAs of pTMB634 and pTMB634 (Fig. 11) were purified and
The fragment containing the merR gene prepared by cutting with mHI and T ₄ −
Ligation was performed using DNA ligase. At that time, 0.5 μg was used on the vector side and 0.5 μg on the inserted DNA side. The obtained hybrid DNA was mixed with CaCl ₂ -treated E. coli DH5α strain at low temperature for transformation. The cells obtained by the transformation were applied to a solid medium containing ampicillin 50 μg / ml and cultured.

After the culturing, the obtained colonies were cultivated again in a liquid medium, and a merR gene-inserted clone having a size of 11.9 kb was selected. The new shuttle vector plasmid prepared from pTMB633 was transformed into pTMZ32 (Fig. 19) and pTMB634.
The new shuttle vector plasmid prepared from
It was named MZ34 (Fig. 20).

From pTMB632 (FIG. 7) in the same manner as above.
Obtaining pTMB635 (Figure 12) and pTMB636 (Figure 13), pTMB635
The new shuttle vector plasmid prepared from
The novel shuttle vector plasmid prepared from MZ36 (Fig. 21) and pTMB636 was named pTMZ38 (Fig. 22).

[0073]

Example 6 In this example, the results of examining the degree of expression of mercury resistance of the vector containing the merR gene of the present invention will be described below.

The degree of development of mercury resistance was examined by the Disc method and the enzyme assay method.

The Disc method is as follows. First, E. coli DH5α strain containing the present invention and various conventional vectors was treated with ampicillin 50 μm.
The cells were cultured overnight in LB medium containing g / ml. After culturing, 200 μl of each culture solution containing these bacterial cells was mixed with 20 ml of LB medium containing 1.5% agar kept at 48 ° C., and this was transferred to a petri dish and solidified. Next, on the solidified plate medium, a disc (Wattman AA Disc with a diameter of 6 m) for antibiotics assay was used.
m) 8 pieces, 0.05, 0.1, 0.2, 0.5, 2.0 respectively
, 5.0, and 10.0 mg / l of HgCl ₂ solution were soaked in 20 μl. These plates were incubated overnight at 37 ° C,
The diameter of the inhibition circle (growth inhibition portion) generated around each Disc was measured. The diameter of the blocking circle is smaller as it has stronger mercury resistance.

As a result of the measurement, it was shown that the vector of the present invention containing the merR gene had a smaller blocking circle and stronger resistance than the blocking circle of the conventional vector containing only the merC-merA gene.

On the other hand, the enzyme assay method was carried out by using E. coli DH5α strain containing various vectors similar to the Disc method and ampicillin.
Incubate overnight in LB medium containing 50 μg / ml and use it in the same medium.
The cells were inoculated with 1/100 inoculum per ml and cultured at 37 ° C. At that time, when the OD at 550 nm was about 0.5,
A mercury solution was added to 10 μg / ml HgCl ₂ and the culture was continued, and a fixed amount of bacterial cells was sampled at regular intervals. The sampled bacterial cells were disrupted in a buffer solution of pH 7.0 using an ultrasonic cell disruptor, and the protein content of the extract was measured by the Lowry method. Also at the same time
The activity of mercury reductase was measured based on the method for measuring the oxidation rate of NADPH, and the activity of mercury reductase per unit protein was calculated.

As a result, those containing the merR gene clearly enhanced mercury resistance in a short time.

[0079]

Example 7 In this example, transformation of T. ferrooxidans carried out according to the electroporation method and mercury vaporization activity of the transformant will be described.

First, T. ferooxidans Y in the logarithmic growth phase
The cells of 4-3 strains were collected from 250 ml of 9K medium, and the bacterial cell pellet was washed to remove iron ions and iron precipitates contained in the medium. Cells. This transformation host cell was electroporated into a buffer (3 mM PIPES, pH = 6.4,
272 mM, Succrose) was suspended in 400 μl (4 ° C.). 5 μg of the shuttle vector plasmid of the present invention, pTMZ32, was mixed with this bacterial cell suspension, and this solution was inserted into a dedicated cuvette with a gap of 0.2 cm.

Then, pulse discharge was performed from a 25 μF capacitor at a voltage of 2500 V through a pulse controller (400 Ω). After discharging, the cells were inoculated into 10 ml of 9K medium and cultured overnight at 30 ° C. After culturing, add 0.25 μg / ml to the cells.
Silica gel plate containing HgCl ₂ for 7-10 days, 30
Incubated at ℃. The transformant T. ferrooxidans was selected from the colonies that appeared on the plate by this culture.

Further, the colonies appearing on the plate were picked up in 5 ml of 9K medium and cultured. After culturing, destroy the proliferated cells and adjust the extract to determine the mercury-dependent NA.
By examining the oxidation of DPH, transformants T. ferroo
The mercury vaporization activity of xidans was investigated.

As a result, the transformant T. ferrooxidans
Had mercury vaporization activity. Also, the plas of the extract
The plasmid in the mid fraction was pTMZ that had been subjected to the introduction treatment.
It was the same as 32.

[0084]

EFFECTS OF THE INVENTION By the development of the present invention, T. ferrooxidans
The ability of the vector having the mercury-resistant selection marker to express the resistance to mercury was remarkably enhanced, and the transformation could be performed more efficiently. In addition, the DNA of the present invention
The advent of factors and host cells made it possible to remain stable in the vector plasmid.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a shuttle vector plasmid prepared according to the present invention.

FIG. 2 is a diagram showing another example of a shuttle vector plasmid produced according to the present invention.

FIG. 3 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 4 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 5 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 6 is a diagram showing an example of a shuttle vector plasmid prepared by a conventional method.

FIG. 7 is a diagram showing another example of a shuttle vector plasmid produced by a conventional method.

FIG. 8 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 9 is a diagram showing still another example of a shuttle vector plasmid produced by a conventional method.

FIG. 10 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 11 is a diagram showing still another example of a shuttle vector plasmid produced by a conventional method.

FIG. 12 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 13 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 14 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 15 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 16 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 17 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 18 is a diagram showing yet another example of a shuttle vector plasmid produced by a conventional method.

FIG. 19 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 20 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 21 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 22 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 23 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 24 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 25 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 26 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 27 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 28 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 29 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 30 is a diagram showing yet another example of a shuttle vector plasmid produced according to the present invention.

FIG. 31: merR of the plasmids of FIGS. 1, 2 and 3
It is a figure which shows a gene part.

FIG. 32 is a diagram showing the merR gene portion of the plasmid of FIG.

FIG. 33 is a diagram showing the merR gene portion of the plasmid of FIG. 5.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1:01 1:19) (72) Inventor Satoshi Takeshima 1-8-2 Marunouchi, Chiyoda-ku, Tokyo No. Dowa Mining Co., Ltd. (72) Inventor Masahiko Numata 1-8-2 Marunouchi, Chiyoda-ku, Tokyo Within Dowa Mining Co., Ltd. (72) Inventor Tomonobu Kusano 2-4-19 Nishigata, Minami-Akita-gun, Akita-gun (72) ) Inventor Kazuyuki Sugawara 2-4-13 Higashi, Ogata-mura, Minami-Akita-gun, Akita Prefecture

Claims (5)

(57) [Claims] 1. A code for a mercury resistance-regulating gene of Thiobacillus having an amino acid sequence shown in Chemical formula 1, Chemical formula 2 or Chemical formula 3, merR (1), merR (2) or merR (3). DNA fragment to be used. Embedded image Embedded image Embedded image 2. A formula 4, the nucleotide sequence shown in Chemical formula 5 and formula 6
Of these , merR (1) ( base number 90 to 567 ), me
rR (2) (base number 117 to 522) or merR
(3) (portion of base numbers 117 to 522) , The DNA fragment according to claim 1. 3. A novel plasmid comprising the Escherichia coli-derived plasmid in which the mercury resistance regulatory gene fragment of Thiobacillus genus according to claim 1 or 2 is incorporated. 4. The origin of replication of a plasmid derived from the genus Thiobacillus, the origin of replication of a plasmid derived from Escherichia coli, or the origin of replication of a broad host range vector belonging to the incompatibility group Q, and the method according to claim 1 or 2. A shuttle vector plasmid containing the mercury resistance-regulating gene fragment of Escherichia coli, a gene of a protein that regulates mercury transport between the outside world and cells, and a group of mercury resistance genes of the genus Thiobacillus consisting of a mercury reductase gene. 5. Mercury-sensitive Thiovachis obtained by transforming mercury-sensitive Thiobacillus ferrooxidans cells with the shuttle vector plasmid according to claim 4.
Mercury resistant transformant of Rus ferrooxidans .