JP4386254B2 - Insertion sequences that function in coryneform bacteria - Google Patents

Description

  The present invention relates to an insertion sequence (IS) having a novel DNA base sequence derived from coryneform bacteria.

Transposition factors such as transposons and insertion sequences have been found in prokaryotic and eukaryotic cells. In addition, insertion sequences of microorganisms are widely studied in E. coli-derived, Shigella-derived, acetic acid-derived, mycoplasma-derived and the like (see Non-Patent Documents 1 to 4).
An insertion sequence, which is one of translocation sequences in microorganisms, has several characteristics. For example, the inserted sequence has a size of about 1 to 2 kb, and inverted repeats [inverted repeat (sequence); IR] exist at both ends. In addition, when an insertion sequence is inserted into a microbial chromosome, an overlapping sequence occurs in the target DNA base sequence (translocation site).

  In recent years, insertion sequences and transposons, which are transposable elements, have been reported one after another in coryneform bacteria. Specifically, IS3181, ISCg1, IS13869, IS719, IS714, IS1206, IS903, ISSCg2, IS1513, IS1253, IS1249, IS1132, IS256, and IS256 belonging to the IS30 family (Patent Documents 1 to 3). 5, refer nonpatent literature 5-11).

As a method for isolating transposable elements such as an insertion sequence derived from a microorganism, a method using a sacB gene product derived from Bacillus subtilis is known (see Non-Patent Documents 12 and 13).
That is, since the sacB gene product is induced and produced by sucrose in the medium, when E. coli having this gene is cultured in a medium containing sucrose, the expression of this gene causes lysis of E. coli and inhibits growth. It is known (see Non-Patent Document 14).
Therefore, when E. coli is transformed with a plasmid having the sacB gene and the plasmid is extracted from E. coli that can grow on a plate containing sucrose, the sacB gene on this plasmid is deleted or inserted (inactivated). What is happening is obtained. A transposable element can be isolated from the one having this insertion mutation (inactivation).

  Also in coryneform bacteria, the sacB gene is known to cause growth inhibition (see Non-Patent Document 13).

With respect to microorganisms, the analysis of whole genomes of 100 strains or more has already been completed, and in recent years, the whole genome sequences of coryneform bacteria have also been determined (see Non-Patent Documents 15 to 17).
Now that we have entered the post-genomic era where expression functions from genomic information are important, in industrially important microorganisms such as coryneform bacteria, a large number of genes are required to perform more effective and efficient gene recombination. Introduction methods, multiple gene deletion methods, and regenerative operations such as mutation, deletion, and introduction to repeated chromosomal DNA are becoming increasingly important.

In the case of a transformation method using an insertion sequence, if the same type of insertion sequence is used for mutation, deletion or introduction after the second time, the inserted sequence introduced the first time is translocated again on the chromosome because the control mechanism is the same. There is a possibility that stable chromosomal mutations, deletions or introduced strains cannot be obtained. Therefore, it is necessary to use insertion sequences having different control mechanisms.
JP-A-7-107976 JP-A-9-70291 JP-A-9-70291 (JP-A-9-70291) European Patent Application Publication No. 0525558 Mol. Jen. Genet (Mol. Gen. Genet), 1973, 122, p. 267-277 Journal of Bacteriol, 1990, 172, p. 4090-4099 Mol. Microbiol, 1993, 9, 211-218 Mol. Microbiol, 1993, Vol. 7, 577-584 Vertes A. A. (Vertes, AA), 4 others, Mole. Microbiol, 1994, Vol. 11, p. 739-746 Jaguar, double. (Jager, W.), three others, FEMS Microbiol Lett., 1995, vol. 126, p. 1-6 Gene, 1996, 170, p. 91-94 Mol. Microbiol, 1994, 11, 738-748 Mol. Jen. Genet, 1999, Vol. 262, p. 568-578 Mol. Jen. Gen. Genet, 2000, 263, p. 1-11 Plasmid, 1995, 34, p. 119-131 Journal of Bacteriology, 1985, 164, p. 918-921 Journal of Bacteriol., 1992, 174, p. 5462-5465 Mol. Jen. Genet (Mol. Gen. Genet), 1983, Vol. 191, p. 138-144 Hiroshi Nonaka, 7 others, “Corynebacterium glutamicum R Genome Analysis”, Abstracts of the 2003 Annual Meeting of the Agricultural Chemical Society of Japan, April 2003, p. 20 Ikeda M. (IkedaM,), one outside, app. Micro viol. Biotechnol. (Appl Microbiol Biotechnol.), 2003, Vol. 62, p. 99-109 Nishio Wai. (Nishio Y), 10 others, Genome Res., 2003, Vol. 13, p. 1572-1579

An object of the present invention is to isolate a novel insertion sequence that enables mutation or introduction into a chromosome by a control mechanism different from that of a known insertion sequence. Another object of the present invention is to create a plasmid incorporating a novel insertion sequence, or to produce a transformant of coryneform bacteria.
In the present invention, the “insertion sequence” means a DNA base sequence that exists on the coryneform bacterial chromosome and can be translocated onto the chromosome or plasmid.

As a result of intensive studies, the present inventors succeeded in isolating a novel insertion sequence having the sequence shown in SEQ ID NO: 1 which is one of translocation factors by a method using the sacB gene, Further research has been conducted and the present invention has been completed.
That is, the present invention
(1) an insertion sequence characterized by having the DNA base sequence shown in SEQ ID NO: 1 that functions in coryneform bacteria,
(2) The insertion sequence according to (1) above, which comprises at least one drug resistance gene in the insertion sequence,
(3) The inserted sequence according to (1) or (2) above, which is incorporated into a plasmid for the purpose of translocation and amplification on a chromosome, and (4) Corynebacterium glutamicum R Strain (FERM P-18976), ATCC13032 strain or ATCC31831 strain, the insertion sequence according to any one of the above (1) to (3),
About.

The insertion sequence of the present invention is characterized by having a DNA base sequence represented by SEQ ID NO: 1 in the sequence listing described later derived from Corynebacterium glutamicum.
The insertion sequence of the present invention has no homology at the DNA level with the insertion sequence previously found from coryneform bacteria, and also exists in a known insertion sequence that functions in coryneform bacteria. It is a novel one that has no homology to genes (amino acid level) and inverted repeats (DNA level).
The insertion sequence of the present invention has so far been described for several coryneform bacteria, such as Corynebacterium glutamicum R strain (FERM P-18976), ATCC13032 strain and ATCC31831 strain, as shown in the Examples below. It is different from the insertion sequence that has been found.

  An example of the basic operation for preparing the insertion sequence of the present invention from the coryneform bacterium that is the source microorganism is as follows. That is, the insertion sequence of the present invention is present on the chromosome of the above-mentioned coryneform bacterium, for example, Corynebacterium glutamicum ATCC 14999 strain, and this strain has a sacB gene derived from Bacillus subtilis and can be replicated in the coryneform bacterium. It can be isolated and obtained by transforming with a plasmid such as plasmid pMV5, smearing on a plate containing sucrose, and extracting the plasmid from the growing sucrose resistant strain.

More specifically, first, chromosomal DNA was extracted from a culture of Bacillus subtilis, for example, the Marburg strain (Biochem. Biophys. Res. Commun., Vol. 119, p. 795-800 (1984)). To do. Using this chromosomal DNA as a template, a region containing known sacB and sacR (sacB gene expression control gene) genes is amplified and isolated by PCR. The amplified DNA fragment is introduced into an E. coli / coryneform bacterium shuttle vector, and Escherichia coli JM109 (commercially available from Takara Shuzo) is transformed using this vector to obtain a transformant.
Subsequently, plasmid DNA is extracted from the obtained transformant, and a coryneform bacterium such as Corynebacterium glutamicum ATCC 14999 is transformed using this plasmid, and the sucrose resistant strain that grows on a plate containing sucrose. Extract the plasmid. Among these plasmids, a plasmid having a size larger than that of the original plasmid is selected, and a DNA fragment inserted into the sacB and sacR gene regions of this plasmid is isolated to separate and acquire the inserted sequence. be able to.

As one of the insertion sequences thus obtained, there can be mentioned a fragment which is present on the chromosomal DNA of the coryneform bacterium, for example, Corynebacterium glutamicum ATCC 14999, and has a size of about 1.2 kb.
The number of recognition sites and the size of the cleaved fragments when the inserted sequence of about 1.2 kb is cleaved with various restriction enzymes are shown in Table 1 below.

In Table 1 above, “recognition site number” by restriction enzyme means that a DNA fragment or plasmid is completely digested with restriction enzyme, and those digests are subjected to 1% agarose gel electrophoresis and 5% polyacrylamide gel according to a method known per se. A value determined from the number of fragments that were subjected to electrophoresis and separable was adopted.
The “size of cleaved DNA fragment” and “size of cleaved DNA of plasmid” are obtained by cleaving lambda phage DNA of E. coli with restriction enzyme HindIII when using agarose gel electrophoresis. Based on the standard line drawn by the migration distance of DNA fragments of known molecular weight on the same agarose gel, and when using polyacrylamide gel electrophoresis, the DNA of E. coli 174 phage (φx174phage) is restricted. The size of each DNA fragment of the cleaved DNA fragment and the plasmid is calculated based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment of known molecular weight obtained by cleaving with the enzyme HaeIII.
In determining the size of each DNA fragment, the result obtained by 1% agarose gel electrophoresis was adopted for the size of fragments of 1 kb or more, and the size of fragments of about 0.1 kb to less than 1 kb was used. By adopting the results obtained by 5% polyacrylamide gel electrophoresis, a more accurate fragment size can be determined.
The size of the plasmid can be determined by adding the sizes of each of the cleaved fragments obtained as described above.

  On the other hand, the nucleotide sequence of the insert sequence derived from the chromosomal DNA of Corynebacterium glutamicum ATCC 14999 is the dideoxynucleotide termination method using the plasmid pUC118 or pUC119 (Takara Shuzo) [dideoxy chain termination method, Sanger, F. et. Al. , Proc. Natl. Acad. Sci. USA, Vol. 74, p.5463 (1977)]. The inserted sequence having the base sequence of the DNA fragment of about 1.2 kb determined as described above has the sequence shown in SEQ ID NO: 1 in the sequence listing described later.

  The insertion sequence of the present invention including the above-described base sequence is not limited to those isolated from natural coryneform bacterial chromosomal DNA, but is also a commonly used DNA synthesizer, such as 394 DNA manufactured by Applied Biosystems. It may be synthesized using an / RNA synthesizer.

  In addition, as described above, the insertion sequence of the present invention obtained from the chromosomal DNA of Corynebacterium glutamicum ATCC 14999 may have a part of the base sequence other than that unless the function of the insertion sequence is substantially impaired. A base may be substituted or deleted, a new base may be inserted, and a part of the base sequence may be rearranged, and these substitutions and deletions Any of the inserted or rearranged base sequences is included in the inserted sequence of the present invention.

  As a plasmid having a sacB gene derived from Bacillus subtilis used for isolation of the insertion sequence of the present invention and capable of replicating in a coryneform bacterium, for example, plasmid pMV5 (Vertes, AA, Inui, M., Kobayashi, M., Kurusu , Y. and Yukawa, H .: Mol. Microbiol., Vol. 11, p.739-746 (1994)) can be used advantageously, but if it is a plasmid having a similar function, it is limited to the above pMV5. It is not a thing. As a shuttle vector for E. coli / coryneform bacteria into which sacB and sacR gene regions can be introduced in addition to pMV5, for example, pCR1 (Kotrba, P., Inui, M., Yukawa, H .: Biochem Biophys Res Commun, No. 1) 289, p.1307-1313 (2001)), pAM330 described in JP-A-58-76779; pHM1519 described in JP-A-58-77895; described in JP-A-58-192900. PAJ655, pAJ611 and pAJ1844; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799; The plasmid pCRY30 described in JP-A-3-210184; the plasmid pCRY2 described in JP-A-2-276575 , PCRY2KE, pCRY2KX, pCRY31, pCRY3KE and PCRY3KX; can be used plasmid pCRY2 and pCRY3 like described in JP-A-1-191686.

  Examples of host coryneform bacteria that have the sacB gene derived from Bacillus subtilis and can be transformed with a plasmid that can replicate and propagate in coryneform bacteria include Corynebacterium glutamicum ATCC 14999, Brevibacterium lactofermentum (Brevibacterium lactofermentum). ATCC 13869; Brevibacterium flavum MJ-233 (FERM BP-1497) and its derived strains; Brevibacterium ammoniagenes ATCC6871, ATCC 13745, ATCC 13746; Brevibacterium divaricatum ATCC 14020 Etc. can be used suitably. Among these coryneform bacteria, Corynebacterium glutamicum ATCC 14999 is most preferably used as a host for the isolation of the insertion sequence of the present invention.

  Transformation of the host coryneform bacterium with the recombinant plasmid may be performed by a method known per se, such as Calvin, NM and Hanawalt, PC, Journal of Bacteriology, 170, p.2796 (1988); Ito, K. , Nishida, T. and Izaki. K., Agricultural and Biological Chemistry, Vol. 52, p.293 (1988), etc., for example, applying a pulse wave to a host microorganism [Satoh, Y. et al. al., Journal of Industrial Microbiology, Vol. 5, p. 159 (1990)].

  The transformant thus obtained is smeared and cultured on a plate containing 2 to 15% sucrose as described above, and each sucrose resistant strain grown is liquid cultured. Next, the plasmid is extracted from the culture by a commonly used method known per se, for example, alkali-SDS method. The extracted plasmid is analyzed with an appropriate restriction enzyme, and a plasmid having a larger size than the original plasmid is selected. By isolating the DNA fragment inserted into the sacB and sacR gene regions of the selected plasmid, the inserted sequence of the present invention can be separated and obtained.

  The transformant can be cultured in a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, etc., and sucrose is preferred as the carbon source. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination. Moreover, as inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate etc. can be used, for example. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be appropriately added to the medium.

The culture can be usually performed at a temperature of about 25 ° C. to about 35 ° C. under aerobic conditions such as aeration stirring and shaking (in the case of liquid culture). The pH during the culture can be around 7 to 8, and the pH during the culture can be adjusted by adding an acid or an alkali. The carbon source concentration at the start of the culture is 5 to 12% by volume.
The culture thus obtained is smeared on a plate containing 2 to 15% sucrose as described above and cultured at 33 ° C. for 1 to 3 days to obtain a sucrose resistant strain into which the inserted sequence has been introduced. it can.

The insert sequence of the present invention is, for example, a DNA fragment that originally existed on the coryneform bacterial chromosome and did not exist on the plasmid used for transformation. This can be proved by observing the phenomenon of translocation from the chromosome to the plasmid). Conversely, the function (transformation function) of the insertion sequence of the present invention can be further confirmed by translocating the inserted sequence translocated on the plasmid onto the chromosome.
Therefore, the insertion sequence of the present invention enables transposon translocation onto the chromosome by a mutually independent control mechanism. As a result, transformation techniques such as multiple gene recombination can be carried out effectively and efficiently.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.

Isolation of the insertion sequence from Corynebacterium glutamicum:
(A) Isolation of sacB resistant strain Plasmid pMV5 (Vertes, AA, Inui, M., Kobayashi, M., Kurusu, Y. and Yukawa, H .: Mol. Microbiol., Vol. 11, p. 739-746 (1994)) was introduced into coryneform bacteria, and the cells were transformed. The host coryneform bacterium used is Corynebacterium glutamicum ATCC 14999. An electric pulse method was used for transformation. 100 mL of the above-mentioned coryneform bacterium (composition: urea 2 g, ammonium sulfate 7 g, monopotassium phosphate 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate 0.5 g, manganese sulfate 4- 6 hydrate 6 mg, iron sulfate 7 hydrate 6 mg, yeast extract 1 g, casamino acid 1 g, biotin 200 μg, thiamine hydrochloride 100 μg dissolved in deionized water to 1 liter (pH 7.4)] After culturing to the initial stage, penicillin G was added to 1 unit / mL, and further cultured with shaking for 2 hours. The cultured cells were collected by centrifugation and washed with 20 mL of a pulse solution [composition: 272 mM sucrose, 7 mM potassium dihydrogen phosphate, 1 mM magnesium chloride; pH 7.4]. Again, the cells were collected by centrifugation, suspended in 5 mL of the pulse solution, 0.75 mL of cells and 2 μL (1 μg) of the above plasmid pMV5 solution were mixed, and allowed to stand in ice for 20 minutes. Using Gene Pulser (manufactured by Bio-Rad), it was set to 2500 volts 25 μF, and after applying a pulse, it was allowed to stand in ice for 20 minutes. The whole amount was cultured at 30 ° C. for 1 hour, and then smeared on an A agar medium containing 10% sucrose and kanamycin 50 μg / mL. The cells were cultured at 30 ° C. for 2 days.

(B) Isolation of Insertion Sequence A plasmid was prepared from the emerging sucrose and kanamycin resistant strains by the method described in JP-A-1-95785. This plasmid was digested with various restriction enzymes SmaI and XbaI, and its size was confirmed. As a result, it was found that the plasmid pMV5 used for control transformation, 1.9 kb, was detected at 1.9 kb, but a 3.1 kb band was detected in 2 strains, and there was a plasmid about 1.2 kb larger. . It was found that the 1.9 kb SmaI / XbaI gene fragment of pMV5 is present in the sacB gene, and the inserted sequence having a size of about 1.2 kb inactivates the sacB gene product. .

Determination of the nucleotide sequence of the insert derived from Corynebacterium glutamicum:
In order to specify the inserted sequence having a size of about 1.2 kb obtained in Example 1, the base sequence was determined by the dideoxynucleotide method using pUC118 or pUC119 (Takara Shuzo). As a result, the sequence described in SEQ ID NO: 1 was revealed. On this sequence, there was an open reading frame (ORF) consisting of 343 amino acids. Also, 22 bp at both ends of this insertion sequence
5 'side IR1; 5'-TAGCTCCCCCAAAACAAAAGCT-3'
3 'side IR2; 5'-TAGCTCTCCCAAATCAAAAGCT-3'
There was an inverted repeat (IR) consisting of and a direct repeat of 2 bp (5′-TA-3) which is the target sequence. The number of recognition sites and the size of the cleaved fragment when the inserted sequence having a size of about 1.2 kb was cleaved with various restriction enzymes were as shown in Table 1 above. The restriction enzyme cleavage point map is shown in FIG.

Presence of Insertion Sequence and Confirmation of Transposition Function (A) Confirmation of Presence of Insertion Sequence Southern hybridization was performed on the chromosomal DNA of Corynebacterium glutamicum ATCC 14999 using the isolated insertion sequence as a probe. The probe was prepared using a random labeling kit (commercially available from Takara Shuzo) using the insertion sequence of about 1.2 kb obtained in Example 1. Southern hybridization was performed according to a conventional method [“Molecular Cloning”, Cold Spring Harbor Laboratory Press (1989)]. As a control, chromosomal DNA of Corynebacterium glutamicum R strain (FERM P-18976) was used. As a result, the inserted sequence hybridized with a chromosome derived from Corynebacterium glutamicum ATCC 14999, but was not hybridized with a chromosomal DNA derived from Corynebacterium glutamicum R strain (FERM P-18976). That is, the insertion sequence of the present invention is not present in the chromosome of the control Corynebacterium glutamicum R strain, but is present in the Corynebacterium glutamicum ATCC 14999 chromosome and can be translocated onto the plasmid from the chromosome. It is shown that.

(B) Preparation of a transposition function detection plasmid The oligonucleotide obtained above was inserted into the oligonucleotide having a size of about 1.2 kb based on the determined sequence and complementary to both ends and having an EcoRV site (Table 2) was synthesized and amplified by the PCR method.

Restriction enzyme EcoRV 5 unit was added to the obtained 1.2 kb PCR fragment, reacted at 37 ° C. for 1 hour, completely digested and treated at 65 ° C. for 15 minutes to inactivate the enzyme. On the other hand, plasmid pHSG398 (Takara Shuzo) was digested completely with HindIII 5 unit, subjected to blunt end treatment with DNA Blunting Kit (Takara Shuzo), mixed with the EcoRV 1.2 kb PCR fragment, 50 mM Tris buffer (pH 7.6), Each component of 10 mM dithiothreitol, 1 mM adenosine triphosphate (ATP), 10 mM magnesium chloride and 1 unit of T4 DNA ligase was added (the concentration of each component is the final concentration) and reacted at 4 ° C. for 15 hours to bind. . The resulting plasmid mixture was used to transform Escherichia coli JM109 (commercially available from Takara Shuzo) by the calcium chloride method [J. Mol. Biol., Vol. 53, p.159 (1970)], and chloramphenicol. A medium containing 50 mg [tryptone 10 g, yeast extract 5 g, sodium chloride 5 g, and agar 16 g dissolved in 1 liter of distilled water] was smeared. The growing strain on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was treated with a restriction enzyme to confirm the inserted fragment. As a result, in addition to the DNA fragment having a size of plasmid pHSG398 of 2.2 kb, an inserted fragment having a size of about 1.2 kb was observed. Next, the constructed plasmid is cleaved with a restriction enzyme HindIII that cleaves this plasmid at one place, a kanamycin resistance gene cassette (Pharmacia) is inserted, and the plasmid is resistant to both chloramphenicol and kanamycin and inserted. A plasmid pHSG398-IS14999-Km having the sequence was prepared.
In this plasmid, the HindIII site is present near the end of the insertion sequence of about 1.2 kb and does not destroy the open reading frame (ORF) encoded by the DNA fragment of about 1.2 kb. Therefore, when this plasmid that does not have a replication region that can grow in coryneform bacteria is transformed into coryneform bacteria and a transformant is obtained using kanamycin resistance as an index, this plasmid is transformed into the coryneform bacterial chromosome. An inserted strain is obtained. Among these, in the chloramphenicol sensitive strain, it can be determined that only the inserted sequence portion is translocated onto the chromosome and the entire pHSG398-IS14999-Km is not inserted, and the inserted sequence obtained in the present invention is Shows that dislocation is possible up.

(C) Confirmation of translocation Plasmid pHSG398-IS14999-Km was introduced into coryneform bacteria, and the cells were transformed. The host coryneform bacterium used is Corynebacterium glutamicum R strain (FERM P-18976) which does not have this insertion sequence. The electric pulse method described above was used for transformation. 50 kanamycin resistant strains were obtained. Of these, only 4 strains were resistant to chloramphenicol, but the rest were chloramphenicol sensitive. This indicates that the insert sequence present on the plasmid pHSG398-IS14999-Km has been translocated onto the chromosome together with the kanamycin resistance gene.

  Since the insertion sequence of the present invention can be translocated and inserted into various sites on the chromosome, it can be used as an effective means for gene analysis. It can also be used for the production of insertion mutants, gene mapping, promoter search, insertion of genetic information, destruction of specific genes, etc., and is industrially useful.

It is a figure which shows the restriction enzyme cutting | disconnection site | part map of the insertion sequence of this invention.

Claims (5)

Insertion sequence polynucleotide, comprising the DNA base sequence shown in SEQ ID NO: 1 that functions in coryneform bacteria. The insert sequence polynucleotide according to claim 1, wherein the insert sequence contains at least one drug resistance gene. The inserted sequence polynucleotide according to claim 1 or 2, which is incorporated into a plasmid for the purpose of translocation and amplification on a chromosome. The insertion sequence polynucleotide according to any one of claims 1 to 3, which is used for transformation of Corynebacterium glutamicum R strain (FERM P-18976), ATCC 13032 strain or ATCC 31831 strain. The insertion sequence polynucleotide according to any one of claims 1 to 4, which is derived from Corynebacterium glutamicum ATCC 14999 strain .

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