JP2005110597A - New plasmid derived from acetic acid bacterium, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plasmid vector having a high gene-introducing efficiency to an acetic acid bacterium, especially the acetic acid bacterium belonging to the genus Acetobacter or the genus Gluconacetobacter, and exhibiting excellent stability in a cell. <P>SOLUTION: The plasmid pGI1 comprises a specific base sequence or a base sequence in which one or several bases are substituted, deleted, inserted, added or inverted in the specific base sequence, and can carry out the autonomous replication in the acetic acid bacterium. The vector for the acetic acid bacterium contains at least a part of the plasmid and a selective marker gene, and can be replicated in the cell of the acetic acid bacterium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to genetic engineering techniques, and more particularly to plasmids useful for genetic manipulation of acetic acid bacteria, in particular, Acetobacter and Gluconacetobacter, and use thereof.

Acetic acid bacteria are alcoholic microorganisms that are widely used in vinegar production. In particular, Acetobacter and Glucon Acetobacter acetic acid bacteria having alcohol oxidizing ability are used for industrial acetic acid fermentation.
Among these, Gluconacetobacter entanii, which is an acetic acid bacterium belonging to the genus Gluconacetobacter, particularly Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) (FERM BP-491 in the Patent Organism Depositary Center Is the most acetic acid bacterium having the highest acetic acid tolerance and capable of accumulating 20% or more of acetic acid in the medium.

On the other hand, in acetic acid fermentation, it is required to develop an acetic acid bacterium that does not decrease the growth ability and fermentation ability even at a higher acetic acid concentration, that is, has high acetic acid resistance. As one means, attempts have been made to clone a gene involved in acetic acid resistance (acetic acid resistant gene) and to breed and improve acetic acid bacteria using the acetic acid resistant gene.
Here, in order to perform genetic manipulation in acetic acid bacteria, a vector capable of autonomous replication in acetic acid bacteria is required. Known multi-copy plasmid vectors used in general genetic manipulation include pUF106 (see, for example, Non-Patent Document 1), pMV24 (see, for example, Non-Patent Document 2), pTA5001 (A), and pTA5001 (B) (for example, (See Patent Document 1).

  However, these multi-copy vectors are prepared by using a plasmid derived from an acetic acid bacterium different from a practical acetic acid bacterium such as Gluconacetobacter enterii including the above-mentioned Acetobacter altoacetigenes MH-24 strain. Therefore, the efficiency of gene introduction into the practical acetic acid bacterium is low. In addition, even if transformation was possible, there were problems such as difficulty in stably maintaining the plasmid vector in the cell, and therefore, it was desired to develop a more practical vector for genetic manipulation in acetic acid bacteria. It was.

"Cellulose, p. 153-158, 1989" “Applied and Environmental Microbiology, Vol. 55, p. 171-176, 1989” Japanese Patent Laid-Open No. 60-9488

  In such a situation, the present invention has a high gene transfer efficiency into acetic acid bacteria, particularly Acetobacter and Glucon Acetobacter acetic acid bacteria having an ability to oxidize alcohol, and is excellent in intracellular stability. For the development of vectors.

Based on the above problems, the present inventor has intensively studied, and as a result, one of the useful acetic acid bacteria, Gluconacetobacter entanii, especially Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) strain. A circular double-stranded DNA plasmid (hereinafter referred to as plasmid pGI1) that can be replicated even in these cells was found.
Furthermore, an E. coli plasmid vector pUC18 having an ampicillin resistance gene as a selectable marker gene was ligated to this plasmid pGI1 to prepare a chimeric plasmid, and transformation into acetic acid bacteria was attempted. Acetobacter altoacetigenes MH-24 strain Acetobacter aceti No. 1023 strain (Acetobacter aceti No.1023) (deposited as FERM BP-2287 at the Patent Biological Depositary Center), Acetobacter aceti IFO3283 strain (Acetobacter aceti IFO3283), and Gluconacetobacter zylinus IFO3288 strain (Gluconacetobacter 288, IFO3288) The selection marker gene was expressed in many acetic acid bacteria, succeeding in obtaining a transformed strain, and completing the present invention.

That is, the present invention described in claim 1 is the plasmid pGI1 shown in the following (A) or (B).
(A) A plasmid comprising the base sequence set forth in SEQ ID NO: 1 in the sequence listing.
(B) The nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing consists of a nucleotide sequence containing substitution, deletion, insertion, addition, or inversion of one or several bases, and can autonomously replicate in acetic acid bacteria. Plasmid.
The present invention described in claim 2 is a vector for acetic acid bacteria, comprising at least a part of the plasmid of claim 1 and a selectable marker gene, and capable of replicating in cells of acetic acid bacteria.

  According to the present invention, a plasmid and a vector for acetic acid bacteria that have high efficiency of gene introduction into acetic acid bacteria and excellent stability in cells are provided. Can do.

Hereinafter, the present invention will be described in detail.
The plasmid pGI1 of the present invention according to claim 1 is shown in the following (A) or (B).
(A) A plasmid comprising the base sequence set forth in SEQ ID NO: 1 in the sequence listing.
(B) The nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing consists of a nucleotide sequence containing substitution, deletion, insertion, addition, or inversion of one or several bases, and can autonomously replicate in acetic acid bacteria. Plasmid.

First, (A) the plasmid consisting of the base sequence described in SEQ ID NO: 1 in the sequence listing is a circular double-stranded plasmid having a length of about 3.1 kbp and having the restriction enzyme map of FIG. In FIG. 1, “Sfi I” and “Hinc II” indicate restriction enzyme recognition sites. The numbers in parentheses indicate the base numbers shown in bp units. Furthermore, “pGI1” in the center indicates the plasmid name, and “3080 bp” indicates the total number of bases of the plasmid.
That is, as shown in FIG. 1, (A) plasmid consists of about 3100 base pairs (bp) described in SEQ ID NO: 1 in the sequence listing, and has three recognition sites for Hinc II and one for Sfi I. It can be obtained as a circular double-stranded DNA plasmid. Moreover, the plasmid (A) does not have recognition sites for EcoR I, Sac I, Kpn I, Sma I, BamH I, Xba I, Sal I, Pst I, Sph I, and Hind III.

Such (A) plasmid is Gluconacetobacter entanii, for example, Acetobacter altoacetigenes MH-24 (deposited as FERM BP-491 in the Patent Organism Depositary). Can be prepared by general genetic engineering techniques.
For example, Acetobacter altoacetigenes MH-24 strain is cultured in an appropriate medium (for example, YPG medium), and then this is collected and lysed. For lysis, a known method for lysing bacteria such as Escherichia coli can be applied. For example, sodium hydroxide or sodium dodecyl sulfate can be used. The target plasmid can be separated and purified from the resulting lysate by a commonly used method such as phenol extraction and cesium chloride density gradient centrifugation in the presence of ethidium bromide.

  (A) Genes essential for replicating or existing in a host cell as a plasmid, such as a gene involved in autonomous replication function of a plasmid and stable maintenance in a host cell, (A) When considered to be carried by a part of the base sequence described in SEQ ID NO: 1 of the sequence listing constituting the plasmid, and base substitution, deletion, insertion, addition, or inversion of this region occurs , May not function as a plasmid. However, in the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing, a region that is not involved in the autonomous replication function of the plasmid or retention in the host cell is deleted or another region is inserted or added. It is considered that a derivative having a reverse position or a reverse position also has a function as a plasmid. Therefore, the plasmid pGI1 of the present invention according to claim 1 is not limited to (A) a plasmid alone, but (B) one or several bases in the base sequence described in SEQ ID NO: 1 in the sequence listing. The plasmid may be a plasmid that comprises a nucleotide sequence that includes substitution, deletion, insertion, addition, or inversion, and that can autonomously replicate in acetic acid bacteria.

  Therefore, in the base sequence described in SEQ ID NO: 1 in the sequence listing in the plasmid (B), the base sequence including substitution, deletion, insertion, addition or inversion of one or several bases is as described above. In the base sequence set forth in SEQ ID NO: 1 in the sequence listing, substitution, deletion, insertion, addition, or reverse of one or several bases in a region not involved in the autonomous replication function of the plasmid or retention in the host cell This means a base sequence containing a position.

In addition, in the plasmid (B), being able to autonomously replicate in acetic acid bacteria means that it can autonomously replicate in acetic acid bacteria cells and can stably exist in the cells.
Any acetic acid bacterium may be used as long as it has an ability to oxidize alcohol, and among them, an acetic acid bacterium belonging to the genus Acetobacter or Glucon acetobacter is preferable.
Examples of acetic acid bacteria belonging to the genus Acetobacter include Acetobacter aceti, and specifically, Acetobacter aceti No. Strain 1023 (Acetobacter aceti No.1023) (deposited as FERM BP-2287 at the Patent Organism Depositary) and Acetobacter aceti IFO 3283 strain (Acetobacter aceti IFO3283).
Examples of acetic acid bacteria belonging to the genus Gluconacetobacter include Gluconacetobacter europaeus DSM6160 strain (Gluconacetobacter europaeus DSM6160) and Gluconacetobacter enteraiii (Gluconacetobacter entanii), for example, Acetobacter altoacetylenes MH-24 strain (Acetobacter altogenes MH-24 -24) (deposited as FERM BP-491 at the Patent Organism Depositary) and Gluconacetobacter zylinus IFO3288 strain (Gluconacetobacter xylinus IFO3288).

  Such a plasmid (B), like the plasmid (A), is a Gluconacetobacter entanii, for example, Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH-24) (Patent Organism Center) To FERM BP-491) by a general genetic engineering technique, specifically, by the procedure and conditions as shown in Example 1.

Such a plasmid pGI1 of the present invention according to claim 1 transforms an acetic acid bacterium as a host, particularly an acetic acid bacterium belonging to the genus Acetobacter and Gluconacetobacter having an ability to oxidize alcohol, by linking a selection marker gene. It can be used as a vector for The present invention according to claim 2 provides such a vector for acetic acid bacteria.
That is, the vector for acetic acid bacteria of the present invention according to claim 2 contains at least a part of the plasmid according to claim 1 and a selectable marker gene, and is capable of replicating in cells of acetic acid bacteria.

  Selectable marker gene means a gene that can be used as a marker to know whether or not the gene responsible for the trait to be introduced has been introduced into acetic acid bacteria. Ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, kanamycin Examples thereof include drug resistance genes such as a resistance gene, a spectinomycin resistance gene, a streptomycin resistance gene, and a tetracycline resistance gene. Specific examples include the ampicillin resistance gene of plasmid pBR322 of Escherichia coli, the kanamycin resistance gene of plasmid pACYC177, the chloramphenicol resistance gene of plasmid pACYC184, and the β-galactosidase gene.

The vector for acetic acid bacteria of the present invention according to claim 2 contains at least a part of the plasmid according to claim 1 and a selectable marker gene. In addition, the gene responsible for the trait to be introduced and the DNA involved in expression regulation It may contain a sequence.
The gene responsible for the trait to be introduced is not particularly limited, and examples include genes for enzymes such as proteases, lipases, DNA synthases, RNA synthases, and enzyme metabolizing enzymes involved in amino acid and organic acid synthesizing systems. Specific examples include alcohol dehydrogenase genes of acetic acid bacteria (see, for example, “Journal of Bacteriology, 175, p. 6857-6866, 1993”).

  The DNA sequence involved in the regulation of expression is not particularly limited as long as it functions efficiently in acetic acid bacteria, in particular, Acetobacter or Gluconacetobacter acetic acid bacteria. Promoters, enhancers, various repressor genes, and The corresponding repressor binding region can be mentioned.

The vector for an acetic acid bacterium of the present invention according to claim 2 may be a plasmid containing the above selection marker gene linked to at least a part of the plasmid pGI1 of the present invention according to claim 1. good.
As a plasmid to be ligated, in addition to a plasmid prepared in advance by introducing a desired gene carrying a trait to be introduced, pUC18 carrying an ampicillin resistance gene, pBR322 carrying a tetracycline resistance gene, pHSG298 carrying a kanamycin resistance gene, etc. And known plasmids. In addition, since the pUC18 plasmid can autonomously replicate in E. coli, an acetic acid bacteria-E. Coli shuttle vector can be obtained by ligating them.

Such a vector for acetic acid bacteria of the present invention according to claim 2 has a selection marker gene, for example, a plasmid containing a selection marker gene (for example, pUC18) as at least a part of the plasmid of the present invention according to claim 1. It can produce by connecting.
Specifically, for example, the plasmid pGI1 of the present invention according to claim 1 is cleaved with a restriction enzyme, amplified by the PCR method and further cleaved with a restriction enzyme, while the plasmid (for example, pUC18) is the same. It can be obtained by cleaving with a restriction enzyme and ligating them with T4 DNA ligase (Ligation).
A wide variety of enzymes can be used as restriction enzymes, and the degree of cleavage is adjusted by adjusting the cleavage reaction time and the like according to the enzyme used. For example, in the case of Sfi I, the enzyme concentration is 1 unit / μg and the temperature is 50 ° C. for 1 hour, and in the case of Aat II, the enzyme concentration is 1 unit / μg and the temperature is 37 ° C. for 1 hour. In the examples described later, Sfi I and Aat II were used.

FIG. 2 shows a preparation procedure and a restriction enzyme map of an example of the vector for acetic acid bacteria of the present invention according to claim 2 (vector pGI18 for acetic acid bacteria obtained by introducing pUC18). In FIG. 2, “Aat
"II" and "Sfi I" indicate restriction enzyme recognition sites. MCS is a multicloning site, Ampr is an ampicillin resistance gene site, and the numbers in parentheses are base numbers in bp units. Further, “pUC18”, “pGI1” and “pGI18” in the center indicate plasmid names, and “2.7 kbp”, “3.1 kbp” and “5.8 kbp” indicate the total number of bases of the plasmid. The vector pGI18 for acetic acid bacteria shown in FIG. 2 contains both pUC18 and pGI1, and the total length is about 5800 base pairs (5.8 kbp).

Such a vector for acetic acid bacteria of the present invention according to claim 2 can be replicated in cells of acetic acid bacteria, in particular, Acetobacter and Gluconacetobacter acetic acid bacteria having an ability to oxidize alcohol. In other words, the vector for acetic acid bacteria of the present invention according to claim 2 can be transformed into acetic acid bacteria to obtain recombinant acetic acid bacteria into which a desired gene has been introduced. Specific examples of acetic acid bacteria are as described in the description of the present invention according to claim 1.
As described above, by ligating a plasmid containing a selectable marker gene (for example, pUC18), an acetic acid bacteria-E. Coli shuttle vector capable of replicating not only in acetic acid bacteria but also in E. coli cells is obtained. Can do.

There is no particular limitation on transformation, and usual procedures and conditions can be applied. For example, the calcium chloride method (see, for example, “Agricultural and Biological Chemistry,” Vol. 49, p. 2091-2097, 1985), electroporation method (see, for example, “Bioscience” -See Bioscience, Biotechnology and Biochemistry, Vol. 58, p. 974-975, 1994 ").
The acetic acid bacterium into which the vector for acetic acid bacteria of the present invention according to claim 2 is introduced is a selective medium corresponding to the phenotype of the vector selection marker gene, for example, if the selection marker gene is a drug resistance gene, It can be selected by culturing in a medium containing the same.

Hereinafter, the present invention will be specifically described by way of examples.
Example 1 (Preparation of plasmid pGI1)
Acetobacter altoacetigenes MH-24 (FERM BP-491), a strain of Gluconacetobacter entanii, YPG supplemented with 6% acetic acid and 4% ethanol The cells were cultured in a medium (containing 3% glucose, 0.5% yeast extract, 0.2% polypeptone) at 30 ° C. for 240 to 336 hours with shaking. The obtained bacterial cells were lysed with sodium hydroxide or sodium dodecyl sulfate, treated with phenol, and further purified with plasmid DNA using ethanol.
The obtained plasmid DNA was cleaved with various restriction enzymes (Takara Shuzo) (37 ° C., enzyme concentration 1 unit / ml), and the base pair length of the obtained DNA fragment was determined by agarose gel electrophoresis. FIG. 1 shows a restriction enzyme map of pGI1. In FIG. 1, the numbers in parentheses indicate the base numbers shown in bp units.

As is apparent from FIG. 1, the obtained plasmid DNA is a circular double-stranded DNA plasmid having three recognition sites for Hinc II and one for Sfi I. The total length of the plasmid is about 3100 bases. Pair (bp). Moreover, it did not have a recognition site for EcoR I, Sac I, Kpn I, Sma I, BamH I, Xba I, Sal I, Pst I, Sph I, Hind III.
The obtained plasmid DNA was designated as plasmid pGI1 and used in the following examples.

Example 2 (determination of the entire base sequence of pGI1)
pUC18 (Takara Shuzo Co., Ltd .: 2686 bp) was cleaved with Sma I, pGI1 was cleaved with Sfi I, and then blunted with T4 DNA polymerase, and ligated to the plasmid pGI1 obtained in Example 1. The base sequence of pGI1 was determined by the Sanger dideoxy chain termination method. The base sequence was determined for the entire region of both DNA double strands so that all the cutting points overlapped.
As a result, the base sequence described in SEQ ID NO: 1 in the sequence listing was determined.

Example 3 (Granting of ampicillin (Amp) resistance gene to pGI1 and preparation of vector for acetic acid bacteria)
An attempt was made to produce a vector for acetic acid bacteria by the procedure shown in FIG.
The plasmid pGI1 obtained in Example 1 was amplified by the PCR method using KOD-Plus- (manufactured by Toyobo Co., Ltd.) and cut with Aat II.
The PCR method was performed as shown in FIG. That is, the PCR method was carried out under the conditions described below using the plasmid pGI1 prepared in Example 1 as a template and primers 1 and 2 having an Aat II recognition site as primers. The base sequences of Primer 1 and Primer 2 are as shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, respectively.
The PCR conditions were 30 cycles, with 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 3 minutes as one cycle.

On the other hand, pUC18 (manufactured by Takara Shuzo Co., Ltd .: 2686 bp) carrying the ampicillin (Amp) resistance gene was cleaved with Aat II (37 ° C., enzyme concentration 1 unit / ml), and the plasmid pGI1 obtained in Example 1 was transformed with T4 DNA ligase Ligation was performed. The reaction solution after ligation was transformed into Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) according to a conventional method, and on an LB medium (tryptone 10 g, yeast extract 5 g, NaCl 5 g / 1 liter) containing 100 μg / ml ampicillin sodium. Thus, an Amp resistant transformant was obtained.
A plasmid was prepared from the obtained transformant, and its restriction enzyme cleavage pattern was analyzed. FIG. 2 shows a restriction enzyme map of the obtained plasmid. In FIG. 2, “Aat II” and “Sfi I” indicate restriction enzyme recognition sites. MCS is a multicloning site, Ampr is an ampicillin resistance gene site, and the numbers in parentheses are base numbers in bp units. Further, “pUC18”, “pGI1” and “pGI18” in the center indicate plasmid names, and “2.7 kbp”, “3.1 kbp” and “5.8 kbp” indicate the total number of bases of the plasmid.

  As is apparent from FIG. 2, the obtained plasmid contained both pUC18 and pGI1, and the total length was about 5800 base pairs (5.8 kbp). This plasmid was designated as vector pGI18 for acetic acid bacteria.

Example 4 (transformation into acetic acid bacteria Acetobacter aceti No. 1023 strain)
The vector pGI18 for acetic acid bacteria prepared in Example 3 was used as Acetobacter aceti No. The strain 1023 (Acetobacter aceti No. 1023) (FERM BP-2287) was electroporated (for example, “Bioscience, Biotechnology and Biochemistry,” Vol. 58, p. 974-). 975, 1994 "). Transformants were selected on YPG agar medium supplemented with 100 μg / ml ampicillin.
The ampicillin resistant transformant grown on the selective medium was extracted and analyzed by a conventional method, and it was confirmed that the vector pGI18 was retained.

Example 5 (Transformation to Acetobacter aceti IFO 3283 strain)
The vector pGI18 for acetic acid bacteria prepared in Example 3 was applied to an Acetobacter aceti IFO3283 strain by electroporation (for example, “Bioscience, Biotechnology and Biochemistry”). ), 58, p. 974-975, 1994 ”). Transformants were selected on YPG agar supplemented with 100 μg / ml ampicillin and 2% acetic acid.
About the ampicillin resistant transformant grown on the selective medium, the plasmid was extracted and analyzed by a conventional method, and it was confirmed that the vector pGI18 was retained.

Example 6 (Transformation to Glucon Acetobacter zyrinas IFO3288 Strain)
The pGI18 prepared in Example 3 was transferred to the Gluconacetobacter xylinus IFO3288 strain (Gluconacetobacter xylinus IFO3288) by electroporation (for example, “Bioscience, Biotechnology and Biochemistry”, 58 Volume, p. 974-975, 1994 "). Transformants were selected on YPG agar medium supplemented with 100 μg / ml ampicillin.
About the ampicillin resistant transformant grown on the selective medium, the plasmid was extracted and analyzed by a conventional method, and it was confirmed that the vector pGI18 was retained.

Example 7 (Transformation to Glucon Acetobacter enterii)
The pGI18 prepared in Example 3 was obtained from Gluconacetobacter entanii, in particular, Acetobacter altoacetigenes MH-24 (deposited as FERM BP-491 in the Patent Biodeposition Center). The cells were transformed by electroporation (see, for example, “Bioscience, Biotechnology and Biochemistry, Vol. 58, p. 974-975, 1994”). Transformants were selected on a YPG agar medium (soft agar medium with an agar concentration of 0.55%) supplemented with 100 μg / ml ampicillin and 3% acetic acid and 4% ethanol.
About the ampicillin resistant transformant grown on the selective medium, the plasmid was extracted and analyzed by a conventional method, and it was confirmed that the vector pGI18 was retained.
From the above, the plasmid pGI1 of the present invention according to claim 1 is useful as a vector that can be used universally in acetic acid bacteria, in particular, in acetic acid bacteria belonging to the genus Acetobacter and Glucon acetobacter having alcohol oxidizing ability. it is obvious.

  According to the present invention, a plasmid and a vector for acetic acid bacteria, which have high efficiency of gene introduction into acetic acid bacteria and excellent in intracellular stability, can be suitably used for carrying out gene manipulation in acetic acid bacteria. it can.

It is the figure which showed the restriction enzyme map of plasmid pGI1 derived from Glucon Acetobacter enterii (Acetobacter altoacetigenes). It is the figure which showed the construction figure and restriction enzyme map of pGI18. FIG. 4 shows the positions of primers for PCR in Example 3.

Explanation of symbols

In FIG. 1, “Sfi I” and “Hinc II” indicate restriction enzyme recognition sites. The numbers in parentheses indicate the base numbers shown in bp units. Furthermore, “pGI1” in the center indicates the plasmid name, and “3080 bp” indicates the total number of bases of the plasmid.
In FIG. 2, “Aat II” and “Sfi I” indicate restriction enzyme recognition sites. MCS is a multicloning site, Ampr is an ampicillin resistance gene site, and the numbers in parentheses are base numbers in bp units. Further, “pUC18”, “pGI1” and “pGI18” in the center indicate plasmid names, and “2.7 kbp”, “3.1 kbp” and “5.8 kbp” indicate the total number of bases of the plasmid.
In FIG. 3, Primer 1 and Primer 2 indicate primer 1 and primer 2, respectively, and “Sfi I” and “Aat II” indicate restriction enzyme recognition sites.

Claims (2)

Plasmid pGI1 shown in (A) or (B) below.
(A) A plasmid comprising the base sequence set forth in SEQ ID NO: 1 in the sequence listing.
(B) The nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing consists of a nucleotide sequence containing substitution, deletion, insertion, addition, or inversion of one or several bases, and can autonomously replicate in acetic acid bacteria. Plasmid. A vector for an acetic acid bacterium comprising at least a part of the plasmid according to claim 1 and a selection marker gene, and capable of replicating in an acetic acid bacterium cell.

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