WO2020021740A1 - Enzymes participating in extracellular electron transfer and utilization of same - Google Patents

Abstract

It has been considered that metal corrosion arises since hydrogen sulfide produced by sulfate-reducing bacteria takes electrons from iron and thus converts iron into iron sulfide. However, it is known that corrosion proceeds even after the surface of iron has been coated with iron sulfide and thus iron no longer comes into contact with hydrogen sulfide. The cause of this phenomenon had been unknown. The present inventors examined the electron uptake mechanism by precisely analyzing cellular membranes of sulfate-reducing bacteria, which proliferate using iron as an electron source, from the viewpoints of bioinformatics, biochemistry, electrochemistry and microscopic approach. As a result, the present inventors newly found enzymes having different structures from membrane enzymes which have been known as participating in electron withdrawal. The present inventors confirmed that electrons are drawn from electrodes exclusively when these enzymes are expressed in a large amount, and clarified for the first time that iron corrosion is caused by electron withdrawal directly from iron by bacteria due to the action of these enzymes, thereby completing the present invention.

Description

Enzymes involved in extracellular electron transfer and their use

(4) The present invention relates to an enzyme involved in extracellular electron transfer, and a method for preventing corrosion of a metal material targeting the enzyme.

破 断 If an oil mining pipeline breaks due to iron corrosion, it will lead to a major accident such as an oil spill, and attempts have been made to clarify the cause of the corrosion and prevent corrosion efficiently. Conventionally, the cause of corrosion has been thought to be that iron is converted to iron sulfide by depriving electrons of hydrogen sulfide formed by sulfate-reducing bacteria. However, it is known that corrosion progresses even after iron sulfide covers the iron surface and the iron stops contacting with hydrogen sulfide, and the cause was unknown.

Microbial reduction of oxidized sulfur species (OSS), such as SO ₄ ²⁻ , SO ₃ ²⁻ , S ₂ O ₃ ²⁻ , S ⁰ , Sn ²⁻ , along with the oxidation of the organic substrate is caused by organic anoxic anaerobic It is one of the main mechanisms of energy production under the ocean (Non-Patent Documents 1-3). Hydrogen (H ₂ ) formed through radioactive decomposition of water and mineral-water reactions is considered to be the main energy source of organic depleted environments ranging from shallow marine sediments to the deep crust, which accounts for almost half of the ocean. (Non-Patent Documents 4-6). However, it is unclear whether it is sufficient to maintain the bacterial populations H ₂ performs sulfur respiratory underground (Non-Patent Documents 7 and 8). The microbial energy requirements of vegetative bacteria found in underground environments were still unknown (Non-Patent Document 9). From ubiquitous and abundance of sulfur respiratory bacteria in the underground environment, sulfide addition to the rare H ₂ and organic matrix minerals (MnS, FeS, FeS ₂₎ or to use a rich solid substrate, such as an energy source Elucidating how and how to use it, especially considering the significant imbalance between H ₂ (n-μM) and sulphate (several mM) in shallow sediments, It was thought to enhance understanding of ecosystems isolated from the sun's rays.

A model in which marine sulfate-reducing bacteria (SRBs) take up electrons directly from elemental iron was first proposed by Dinh et al. They isolated SRB from marine sediment supplied with iron granules as the sole electron donor. Recent studies also show that direct electron uptake by SRBs via conductive filaments is important for anaerobic oxidation of methane in co-cultures of methane-utilizing archaea in marine sediments and poorly cultured SRBs. (Non-Patent Documents 11 to 13). To such extracellular electron transport potentially solid minerals makes it possible to use as an electron source, electrons uptake from minerals during reduction of microorganisms OSS is, H ₂ and organic-limited Can occur in sediments. However, the biological mechanism and energetics of the proposed electron uptake process were not clear, and the distribution and importance of the electron uptake mechanism in energy-limited marine sediments remained unclear.

The present inventors have analyzed the cell membrane of sulfate-reducing bacteria that grow using iron as an electron source in detail using bioinformatics, biochemistry, electrochemistry, and microscopy to analyze the electron uptake mechanism. As a result, we succeeded in finding a new enzyme group that has a different structure from the membrane enzymes involved in electron extraction that were known so far. The present inventors have confirmed that electrons are extracted from the electrode only when the enzyme is expressed in a large amount, and that iron corrosion is caused by bacteria directly extracting electrons from iron by the action of this enzyme. The present invention has been completed for the first time.

Therefore, the present invention specifically relates to the inventions described in the following (1) to (22):
(1) an amino acid sequence according to one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18; SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 , 16 and 18; an amino acid sequence having at least 80% identity to the amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; An amino acid sequence in which 1 to 20 amino acids have been substituted, deleted, inserted, or added to the amino acid sequence described in one sequence; or SEQ ID NO: 2, 4, 6, 8, 10 , 12, 14, 16, and 18, a cell surface protein having an amino acid sequence having a homology score of 200 or more with the amino acid sequence described in one sequence selected from the group consisting of:
(2) The protein according to (1), which has an extracellular electron transfer effect.
(3) The protein according to (1) or (2), which is a cytochrome enzyme or a β-propeller protein.
(4) A nucleic acid molecule having a base sequence encoding the protein according to any one of (1) to (3).
(5) The base sequence described in one sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17, or SEQ ID NOs: 1, 3, 5, 7, 9, 11, The nucleic acid molecule according to (4), which has a base sequence that binds under stringent conditions to a nucleic acid having a sequence complementary to the base sequence described in one of the sequences selected from the group consisting of, 13, 15, and 17.
(6) A vector comprising the nucleic acid molecule according to (4) or (5).
(7) A host cell having the vector according to (6).
(8) The method according to any one of (1) to (3), including culturing the host cell according to (7) to express the protein according to any one of (1) to (3). The method for producing a protein according to the above.
(9) An antibody or an immunoreactive fragment thereof, or an aptamer, which binds to the protein according to any one of (1) to (3), or a nucleic acid molecule according to (4) or (5) Antisense nucleic acid, dsRNA, or aptamer.
(10) The antibody or immunoreactive fragment, aptamer, or antisense thereof according to (9), which has an effect of inhibiting the extracellular electron transfer activity of the protein according to any one of (1) to (3). Nucleic acid, or dsRNA.
(11) A composition comprising the antibody according to (10) or an immunoreactive fragment thereof, an aptamer, an antisense nucleic acid, or dsRNA.
(12) A method for determining a protein that causes corrosion of a metal material, comprising identifying an electron transfer enzyme from a cell surface protein of a bacterium present on the surface of the metal material.
(13) The method according to (12), wherein the identification of the electron transfer enzyme is performed by determining a protein having an electron transfer reaction center.
(14) The selection of the electron transfer enzyme has 80% or more identity with the amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. (12) The method according to (12), which is performed by selecting a protein having an amino acid sequence.
(15) binding to an antibody that binds to an electron transferase or an immunoreactive fragment or aptamer thereof, or a gene encoding the electron transferase, which is determined as a causative protein for corrosion of metallic materials by the method of (12) to (14) A method for producing a corrosion inhibitor, comprising producing an antisense nucleic acid, dsRNA, or aptamer.
(16) Identifying an electron transferase from a cell surface protein of a bacterium present on the surface of a metal material by the method of (12) to (14), and determining the determined antibody binding to the electron transferase or its immunoreactivity A method for producing a corrosion inhibitor, comprising producing an antisense nucleic acid, dsRNA, or aptamer that binds to a fragment or an aptamer, or a gene encoding the electron transfer enzyme.
(17) A method for preventing corrosion of a metal material, which comprises inhibiting the function or expression of the electron transfer enzyme in bacteria existing on the surface of the metal material.
(18) The method according to (17), wherein the electron transfer enzyme is the enzyme according to any one of claims 1 to 3.
(19) The function or expression of the electron transfer enzyme is inhibited, but the potential of the metal material having a negative potential is reduced to −0.32 V vs. potential. (17) The method according to (17), wherein the method is performed by stopping the metabolism that pulls out electrons of bacteria by setting the potential to a potential higher than SHE.
(20) The method according to (18), comprising contacting the composition according to (11) with a surface of a metal material.
(21) The method according to (17), comprising contacting the antibody or aptamer produced by the production method according to (15) or (16) with a surface of a metal material.
(22) identifying an electron transfer enzyme from bacteria existing on the surface of the metal material; and
A method for preventing corrosion of a metal material, comprising inhibiting the function or expression of an electron transfer enzyme in bacteria existing on the surface of the identified metal material.
(23) A method for preventing corrosion of a metal material installed in an external environment,
Collecting a sample containing bacteria having cell surface enzymes from the external environment;
Identifying cell surface electron transfer enzymes present in the sample;
Preparing an antibody that binds to the enzyme or an immunoreactive fragment or aptamer thereof, or an antisense nucleic acid, dsRNA, or aptamer that binds to a gene encoding the enzyme;
A method for preventing corrosion of a metal material, comprising flowing the antibody, aptamer, antisense nucleic acid, or dsRNA through a pipe.

に お い て In one aspect, the present invention relates to proteins involved in extracellular electron transfer. The protein of the present invention is present in the cell membrane (particularly, the outer membrane (OM)) because it is involved in electron extraction from outside the cell. As used herein, the term “cell surface protein” means that the protein is present in the cell membrane, and may be present outside, inside, or through the cell membrane. That is, the term “cell surface protein” does not necessarily require exposure to the cell surface, but also includes, for example, proteins located on the inner membrane (IM) side of the OM. Further, the cell surface protein may be a protein having at least a signal peptide necessary for localization on the cell surface and / or near the cell membrane.

タ ン パ ク 質 The protein of the present invention has an amino acid sequence described in any one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. When these sequences include a signal sequence, the protein of the present invention may have an amino acid sequence that does not include the signal sequence. Alternatively, the protein of the present invention has an amino acid sequence having 80% or more identity with the amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. May be provided. The amino acid sequence identity refers to the ratio (%) of the number of amino acids of the same type in the range of amino acid sequences to be compared between two types of proteins, for example, using a known program such as BLAST or FASTA. Can be determined. The identity of the above-mentioned protein of the present invention may be greater than 80% or more, for example, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. It may be the same.

Alternatively, the protein of the present invention may have 1 to 20, 1 to 10 amino acids in the amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18. , 1 to 8, 1 to 5, 1 to 3 amino acids may be substituted, deleted, inserted or added.

Alternatively, the protein of the present invention may be an amino acid having a homology score of 200 or more with the amino acid sequence described by Blast in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. It may be an array. The homology score is obtained by aligning the amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 with the amino acid sequence of the target protein, The score of each amino acid to be obtained can be obtained from a score matrix and calculated as a sum thereof (refer to http://www.gsic.techtech.ac.jp/supercon/supercon2004-e/alignmentE.html). For example, the homology score can be determined using a known BLAST program. As the score matrix, BLOSUM62, PAM32, and the like are known, and in this specification, BLOSUM62 is preferable.

タ ン パ ク 質 The protein of the present invention has an extracellular electron transfer effect. As used herein, the term “extracellular electron transfer action” means an action in which a cell having the protein comes into contact with a solid surface capable of donating electrons to directly extract electrons from the solid surface. To determine whether a protein has extracellular electron transfer activity, add cells expressing the protein to a reactor where the electrode is the only electron donor, and measure the cathode current density after 30 minutes to 24 hours. Can be determined. If the cathodic current density increases, the protein is determined to have extracellular electron transfer activity.

Preferably, the protein of the present invention is a cytochrome enzyme, a β-propeller protein, or a porin protein. The cytochrome enzyme is a kind of heme protein containing heme iron having a redox function, and cytochromes a, b, c, d, f, and o are known (Chem. @ Rev. (2014) 114 ( 8): 4366-4469). Therefore, when a certain protein is a protein having a heme binding motif (CXXCH, CXXXCH, CXXXXXCH, or CXXXXXXCH; C is cysteine and H is histidine) and is a protein having electron transfer ability, Can be determined to be. Further, β-propeller protein is a protein having a three-dimensional structure in which four to eight blade-shaped structures made of β-sheets are conically surrounded around a central axis. Whether a certain protein is a β-propeller protein can be determined by three-dimensional structure analysis or three-dimensional structure simulation. Porin protein is a transmembrane protein containing a β-barrel structure with an inverse equilibrium β-sheet. Porin proteins are also known to form complexes with cytochrome proteins and participate in extracellular electron transfer (Marcus @ J. @ Edwards et al., J. Biol.Chem.published @ online @ April @ 10, $ 2018). Whether or not a certain protein is a porin protein can be determined by three-dimensional structure analysis or three-dimensional structure simulation. Preferably, these cytochrome enzymes, β-propeller proteins, or porin proteins are proteins present in the cell membrane (preferably the outer membrane). Further, since the β-propeller protein and / or the porin protein exerts an extracellular electron transfer effect by forming a complex with the cytochrome enzyme, it is preferable that the β-propeller protein and / or the porin protein be complexed with the cytochrome enzyme. Forming the body.

In another aspect, the present invention relates to a nucleic acid molecule having a base sequence encoding the protein. The nucleic acid molecule of the present invention may have another sequence as long as it has a base sequence encoding the protein. As an example, the nucleic acid molecule of the present invention may be a base sequence described in one sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17, or It has a base sequence that binds under stringent conditions to a nucleic acid having a sequence complementary to the base sequence described in one sequence selected from 5, 7, 9, 11, 13, 15, and 17. As used herein, “hybridize under stringent conditions” means to hybridize under hybridization conditions commonly used by those skilled in the art. For example, Molecular Cloning, a Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), or a method described in Current Protocols in Molecular Biology, which can be determined by the method described in Current Biology, Molecular Biology, etc. For example, hybridization conditions are 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0.2 M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). The conditions may be such that hybridization is carried out at 42 ° C. in medium and then washing at 42 ° C. with 0.5 × SSC.

The present invention further relates to a vector containing the nucleic acid molecule. The vector is not particularly limited as long as it is a vector capable of introducing the nucleic acid molecule into a host cell, and may be any plasmid vector, a retrovirus vector, an adenovirus vector, a lentivirus vector, an adenovirus vector, depending on the host cell. A viral vector such as an associated virus (AAV) vector can be used.

The present invention also includes a host cell having the vector. As the host cell, cells suitable for expressing the target protein, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be appropriately selected.

In another aspect, the present invention relates to a substance that inhibits the function or expression of the protein or the nucleic acid molecule (hereinafter, referred to as “inhibitor”). In particular, the invention relates to an antibody or an immunoreactive fragment thereof, or an aptamer, which binds to said protein, or an antisense nucleic acid molecule, dsRNA, or aptamer, which binds to said nucleic acid molecule. These inhibitors preferably have an action of directly or indirectly inhibiting the extracellular electron transfer action of the protein of the present invention. The present invention includes compositions containing such inhibitors, which compositions can be used as metal corrosion inhibitors. Iron can be mentioned as a metal.

腐 食 The corrosion prevention method targeting proteins according to the present invention can prevent iron corrosion and the like without using a chemical having a large effect on the environment such as a bactericide.

D. in OSS FIG. 2 is a diagram showing the distribution of homologous extracellular cytochromes of ferrophilus ΔIS5. (A) shows a phylogenetic tree obtained by 16S @ ribosomal RNA (rRNA) sequence alignment. (B) D. which are encoded by the DFE_450 and DFE_464 genes of E. ferrophilus IS5, and their homologs (identified using the NCBI blastp, max score> 200); sulfurreducens @ PCA's OmcB, OmcE, OmcS and OmcZ, S. 1 shows a protein phylogenetic tree derived from the amino acid sequence of OMC including OmcB and MtrC of oneidensis MR-1 and Cyc2 of Acidithiobacillus ferrooxidans ATCC 23270. b represents bacterium. Scale bars represent estimated sequence variance or amino acid changes. (A) Inner membrane (IM) and outer membrane isolated from lactate-starved cells and stained with Coomassie Brilliant Blue (CBB) and heme-reactive 3,3 ', 5,5'-tetramethylbenzidine-H ₂ O ₂ ( 2 shows the protein profile of the (OM) fraction. In the heme positive bands (indicated by # and *), transcripts of DFE_461 and DFE_449 were detected by LC-MS. Non-specific heme staining detected a minor band corresponding to ferroxidase (DFE — 1154, 14.1 kDa, △). (B) (Left) Lactate starvation (red) and sufficiency (blue) normalized by OM protein concentration. 3 shows an absorption spectrum of an OM fraction extracted from ferrophilus IS5 cells. Solid and dotted lines show spectra measured under reducing and oxidizing conditions, respectively. (Right) A graph showing a comparison of the solelet peak absorption at 419 nm. (C) D. FIG. 4 is a graph showing the expression of seven multi-heme cytochromes and two β-propeller proteins (hatched bars) located in close proximity in the ferrophilus IS5 genome by lactate-starved and lactate-sufficient cells. Intact D. 3 shows the results of in vivo electrochemical measurement using ferrophilus IS5 cells. (A) Graph showing the results of cathodic current versus time measurements performed in an anaerobic reactor with an indium-tin doped oxide electrode maintained at -0.4 V (vs. SHE) as the only electron donor. is there. Lactic acid starvation added to the reactor ferrophilus IS5 cells, lactic acid sufficiency. ferrophilus IS5 cells, H ₂ -consumable. vacuolatum and sterile medium are indicated by red, blue, pink and black lines, respectively. Arrows indicate when cells or sterile media were added to the reactor. (B) shows a linear sweep voltammogram measured after current generation in panel a. (C) the redox potential of outer membrane cytochromes (OMCs) that can produce NADH and / or reduced menaquinone (MQH ₂ ) promotes the reduction of OSS and intermediates such as adenosine phosphosulfate (APS); 3 shows an energy diagram of an extracellular electron uptake model of the IS5 strain. Cyt represents periplasmic C-type cytochrome. Lactate-starved D. 1 shows micrographs of ferrophilus IS5 cells and their nanowires. (A and b) Scanning electron microscope images of cells attached to the electrode surface. The scale bars of a and b represent 10 μm and 500 nm, respectively. (Ce) Transmission electron microscope images of cells stained with cytochrome reactive 3,3′-diaminobenzidine (DAB) -H ₂ O ₂ . (C) shows the results of negative DAB staining in the absence of H ₂ O ₂ . (D and e) Show the results of DAB staining positive by addition of H ₂ O ₂ . The scale bar corresponds to 500 nm for c and d and represents 50 nm for e. The bamboo-like nanowire structure was clearly visible only when DAB staining was positive. (F and g) Fluorescence microscopy images of cells stained with protein-specific NanoOrange (green) and membrane-specific FM 4-64FX (red). The scale bar represents 5 μm. Arrowheads indicate nanowires.

The protein of the present invention can be obtained by culturing a host cell having the vector of the present invention in a medium suitable for the host cell under culture conditions suitable for expression of the protein by the host cell to express the protein. Can be manufactured.

The present invention also relates to a method for determining a protein that causes corrosion of a metal material, including identifying an electron transfer enzyme from a cell surface protein of a bacterium present on the surface of a metal material as a protein that causes corrosion of the metal material. Information on the cell surface protein of the bacterium present on the surface of the metal material can be obtained by culturing the bacterium and determining the base sequence of the gene encoding the protein in the culture. Whether or not a sequenced protein is an electron transfer enzyme can be determined based on whether or not it has an electron transfer reaction center. In the present specification, the electron transfer reaction center is not particularly limited, but includes, for example, a heme binding domain, an iron ion cluster, and the like. Further, the electron transfer enzyme is preferably a cell surface protein or a membrane protein (preferably, an outer membrane protein). Therefore, in one embodiment, the present invention comprises identifying a protein that is a membrane protein and an electron transfer enzyme from a cell surface protein of a bacterium present on the surface of a metal material as a protein that causes corrosion of the metal material. The present invention relates to a method for determining a causative protein of a metal material. Whether or not the candidate protein is a membrane protein (or outer membrane protein) can be analyzed based on the nucleotide sequence information of the obtained gene. As an example, the electron transfer enzyme may be an amino acid sequence described in one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, or Jing @ Liu et al., Chemical Reviews (2014). ) 114: 4366-4469 may be performed by selecting a gene encoding a protein having an amino acid sequence having 80% or more identity with the protein disclosed in J. Am. In particular, it is known that even when some amino acids constituting a protein are substituted with an amino acid having similar properties (charge or parent / hydrophobicity) to the amino acid, its structure and activity are easily maintained. A protein having an amino acid sequence having 80% or more identity can be obtained by performing such "conservative amino acid substitution" on the original amino acid sequence. Where two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence identity or similarity may be adjusted upwards to modify the conservative nature of the substitution (Pearson, {Methods Mol. Biol. # 243: 307-31 (1994)). Such conservative amino acid substitutions include substitutions between amino acids having an aliphatic side chain such as glycine, alanine, valine, leucine, and isoleucine; substitutions between amino acids having an aliphatic hydroxyl side chain such as serine and threonine; Substitution between amino acids having amide-containing side chains such as asparagine and glutamine; substitution between amino acids having aromatic side chains such as phenylalanine, tyrosine, histidine, and tryptophan; and basic side chains such as lysine, arginine, and histidine. Substitution between amino acids having acidic side chains such as aspartic acid and glutamic acid, substitution between amino acids having sulfur-containing side chains such as cysteine and methionine, glycine, asparagine, glutamine, serine, threonine, Substitutions between amino acids with uncharged polar side chains such as syn, cysteine, and tryptophan; substitutions between amino acids with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine; threonine; Substitutions between amino acids having β-branched side chains such as valine and isoleucine are included, for example, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-. Substitution between glutamines can be mentioned.

In another embodiment, the present invention relates to an antibody or an immunoreactive fragment or aptamer thereof, which binds to an electron transferase determined as a causative protein of a metal material by the above method, or a gene encoding the electron transferase. The present invention relates to a method for producing a corrosion inhibitor, which comprises producing an antisense nucleic acid, dsRNA, or an aptamer. Therefore, the present invention provides a method for identifying an electron transferase from a cell surface protein of a bacterium present on the surface of a metal material by the method, and an antibody, an immunoreactive fragment or an aptamer thereof, which binds to the determined electron transferase Or a method for producing a corrosion inhibitor, comprising producing an antisense nucleic acid, dsRNA, or aptamer that binds to a gene encoding the electron transfer enzyme.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Further, the antibody of the present invention may be an antibody of human, mouse, rat, hamster, guinea pig, rabbit, dog, monkey, sheep, goat, camel, chicken, duck, etc., and preferably, hybridoma is produced. The antibody is preferably an animal antibody, more preferably a mouse, rat or rabbit antibody. The immunoglobulin class of the antibody of the present invention is not particularly limited, and may be any immunoglobulin class (isotype) of IgG, IgM, IgA, IgE, IgD, or IgY, and is preferably IgG. When the antibody of the present invention is an IgG, any subclass (IgG1, IgG2, IgG3, or IgG4) may be used. Further, the antibody of the present invention may be monospecific, bispecific (bispecific antibody), or trispecific (trispecific antibody) (for example, WO1991 / 003493). The antibodies of the present invention also include immunoreactive fragments of the antibodies. As used herein, the term "immunoreactive fragment of an antibody" refers to a protein or peptide containing a part (partial fragment) of an antibody, and a protein that retains the effect of the antibody on the antigen (immunoreactivity / binding property). Or F (ab ′) ₂ , Fab ′, Fab, Fab ₃ , single-chain Fv (hereinafter referred to as “scFv”), (tandem) bispecific single-chain Fv (sc (Fv)) ₂ ), single-stranded triple body, nanobody, divergent VHH, pentavalent VHH, minibody, (double-stranded) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity retargeting molecule (DART) ), triabodies (or tribodies), tetrabodies (or [sc _{(Fv) 2]} 2 Or _{(scFv-SA) 4)} disulfide bond Fv (hereinafter, referred to as "dsFv"), compact IgG, can be given a heavy chain antibody, or polymers thereof.

書 As used herein, the term "aptamer" refers to a nucleic acid molecule or peptide that specifically binds to a specific molecule. When the aptamer is a nucleic acid, it may be RNA or DNA. The form of the nucleic acid may be double-stranded or single-stranded. The length of the aptamer is not particularly limited as long as it can specifically bind to the target molecule, and is, for example, 10 to 200 nucleotides, preferably 10 to 100 nucleotides, more preferably 15 to 80 nucleotides, and still more preferably 15 to 50 nucleotides. Of nucleotides. Aptamers can be selected using methods well known to those skilled in the art, and include, for example, the SELEX method (Systematic Evolution \ of \ ligands \ by \ Exponential \ Enrichment) (Turk, C. and \ Gold, L. (1990) Science. 510) can be used.

に お い て In the present specification, “antisense nucleic acid” refers to a nucleic acid molecule having a sequence complementary to a target sequence, and may be DNA or RNA. The antisense nucleic acid does not need to be 100% complementary to the target sequence, and may contain a non-complementary base as long as it can specifically hybridize under the above-mentioned stringent conditions. When introduced into cells, antisense nucleic acids bind to target sequences and inhibit transcription, RNA processing, translation, or stability. In addition, antisense nucleic acids include those having a polynucleotide mimetic and a modified backbone in addition to the antisense polynucleotide. Such an antisense nucleic acid can be appropriately designed and manufactured (for example, chemically synthesized) using a method well known to those skilled in the art based on the target sequence information.

"DsRNA" is a RNA that has a sequence complementary to a target sequence at least in part by RNA interference (RNAi), and degrades the mRNA by binding to the mRNA having the target sequence, thereby degrading the target sequence. RNA containing a double-stranded RNA structure that suppresses translation (expression). The dsRNA includes siRNA (short @ ferring @ RNA) and shRNA (short @ hairpin @ RNA). The dsRNA does not need to have 100% homology with the target sequence as long as it suppresses target gene expression. The dsRNA may be partially substituted with DNA for stabilization or other purposes. The siRNA is preferably a double-stranded RNA having 21 to 23 bases. siRNA can be obtained by methods well known to those skilled in the art, for example, as an analog of chemically synthesized or naturally occurring RNA. shRNA is an RNA short strand having a hairpin turn structure. The shRNA can be obtained by a method well known to those skilled in the art, for example, by chemical synthesis or by introducing a shRNA-encoding gene into a cell and expressing it.

に お い て In another aspect, the present invention relates to a method for preventing corrosion of a metal material, comprising inhibiting the function or expression of an electron transfer enzyme in a bacterium present on the surface of the metal material. The electron transfer enzyme may be the protein of the present invention or a protein appropriately determined by the above method. Inhibition of the function or expression of an electron transferase includes an antibody that binds to the electron transferase or an immunoreactive fragment or aptamer thereof, or an antisense nucleic acid, dsRNA, or aptamer that binds to a gene encoding the electron transferase. This can be performed by bringing the composition into contact with the surface of the metal material. Thus, the present invention provides a method for inhibiting the corrosion of a metal material, which comprises identifying an electron transfer enzyme from bacteria present on the surface of the metal material by the above method, and inhibiting the function or expression of the identified electron transfer enzyme. Including prevention methods. The metallic material may preferably be in the external environment, for example in water, such as fresh water or seawater. Therefore, in another aspect, the present invention relates to a method for preventing corrosion of a metal material installed in an external environment, comprising collecting a sample containing bacteria having cell surface enzymes from the external environment, Identifying an electron transfer enzyme, preparing an antibody that binds to the enzyme or an immunoreactive fragment or aptamer thereof, or preparing an antisense nucleic acid, siRNA, or aptamer that binds to a gene encoding the enzyme; Includes a method for preventing corrosion of metallic materials, comprising flowing an aptamer, antisense nucleic acid, or siRNA through a pipe.

{In still another embodiment, the present invention provides a metal material having a negative potential of -0.32 V {vs. SHE or more (ie, -0.32V@vs. SHE, or a value larger in the positive direction than that, for example, -0.2, -0.1, 0, 0.1, and 0.2V@vs. SHE) Etc.) or -0.32V@vs. The present invention relates to a method for preventing corrosion of a metal material, including inhibiting the function or expression of an electron transfer enzyme in a bacterium existing on the surface of a metal material by stopping the metabolism that pulls out electrons of the bacterium above SHE to stop the metabolism. Here, “V vs.SHE” means an electrode potential based on a standard hydrogen electrode. According to the findings of the present inventors, since the energy of about 0.3 V is the minimum energy required for driving the metabolism to extract electrons, the potential of the negative potential metal material is set to −0. .32V @ vs. SHE or more or -0.32V@vs. By raising it above SHE, it is possible to stop the metabolism that pulls out electrons of bacteria. The potential of the metal material can be measured with a reference electrode. For example, in the case of the inside of a pipe, it can be measured with a silver-silver chloride electrode. In this case, the measurement of the potential is performed with a silver-silver chloride electrode, converted into the value of a standard hydrogen electrode, and V vs. The value of SHE can be obtained. Further, the measurement of the potential can be performed simultaneously while adjusting the potential using a potentiostat. The adjustment of the potential can be appropriately performed using a means capable of applying a voltage to a metal material such as a potentiostat.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to these examples. All documents cited throughout the application are incorporated herein by reference in their entirety.

(Culture of microorganisms)
D. ferrophilus IS5 (DSM No. 15579) was precultured in butyl rubber stoppered vials containing 100 ml of DSMZ195c medium at 28 ° C. under CO ₂ / N ₂ (20:80, v / v) oxygen-free conditions. After 5 days, the culture contained a small amount of black iron sulfide precipitant. A portion of the cell suspension is diluted 10-fold with fresh DSMZ195c medium supplied with 21 or 110 mM lactate, and further cultured under the above conditions for 5 days to produce a lactate-starved state or a lactate-sufficient state, respectively. I let it. C. except that 15 mM fumarate was added as the sole electron donor instead of lactate. Desulfobacterium vacuolatum (JCM 12295) was cultured under the same conditions as in ferrophilus IS5. Shewanella oneidensis MR-1 was obtained by resuspending cells collected from an isolated colony with a platinum loop in an LB medium, culturing aerobically, and then culturing with shaking vigorously at 30 ° C. for 15 hours. The culture was centrifuged at 5000 × g for 10 minutes, the resulting pellet was washed, and at a modified concentration of electron donor and acceptor (60 mM lactate and 1 mM fumarate), Gorby et al. Natl.Acad.Sci.USA 103, 11358-11363 (2006)), and resuspended in 3 ml of an anoxic chemically defined medium. The cell culture (OD 600 nm 1.5) was placed in a 5 ml vial with a butyl rubber stopper under a 100% N ₂ environment and further cultured at 30 ° C. for 10 hours without stirring. 10.0 mM NH ₄ Cl, 1.0 mM K ₂ HPO ₄ , 0.5 mM MgCl ₂ , 0.5 mM CaCl ₂ , 5.0 mM NaHCO ₃ , 10 mM Na ⁺ -HEPES (pH 7.0), 0.5 g yeast extract And Geobacter sulfurreducens PCA (ATCC 51573) were cultured in anoxic PS medium containing 1 mM of 10 mM sodium acetate and 40 mM sodium fumarate as an electron donor and an acceptor, respectively. After 5 days of culture at 30 ° C., the cell culture was further cultured at 24 ° C. for 3 days until reaching stationary phase. Escherichia coli was cultured aerobically by resuspending cells collected from the isolated colonies with a platinum loop in an LB medium, and then cultured at 37 ° C. for 12 hours with vigorous shaking.

(Genome sequence analysis and analysis)
D. Ferrophilus IS5 nucleic acid molecules were extracted from fresh cell pellets using the Nucleobond AXG Column Kit (TakaraBio). For single molecule real-time (SMRT) cell sequencing, a 15 kb insertion library was constructed on a PacBio RSII sequencer (Pacific Bioscience). A total of 57,299 inserts with an average size of 11 kbp were completely sequenced. Using the HGAP protocol performed in the SMRT analysis (version 2.3, Pacific Bioscience), The ferrophilus IS5 genome was constructed. The output generated consisted of two circular contigs. The first contig was 3,702,182 base pairs (bp), the average coverage was 135.36 times, the second contig was 64,963 bp, and the average coverage was 37.77 times. The ends of both contigs were deduplicated and circularized to give a single 3,677,055 bp circular chromosome and a 43,052 bp circular plasmid. Genome annotation was performed with MiGAP (H. Sugawara et al., Paper presented at the 20th Int. Conf. Genome Informatics 1-2, Kanagawa, Japan, 2009). The chromosome had 3,375 RNA coding sequences and 62 tRNAs. The gene, and the six rRNA genes, contained 56 coding sequences in the plasmid. The genome was searched for the heme binding motif CXnCH (n = 2-5) for the identification of heme binding proteins. The intracellular location of the identified heme protein was predicted using Psortb (NY Yu et al., Bioinformatics 26, 1608-1615 (2010)).

(Transcriptome analysis)
Total RNA was prepared using the NucleoSpin RNA kit (TakarBio). RRNA was removed using a Ribo-Zero Magnetic Kit (Gram-negative bacteria) and a nucleic acid molecule library was prepared according to the manufacturer's guidelines for sequencing using the TruSeq Stranded mRNA Library Prep Kit (Illumina). RNA quality was confirmed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The c-nucleic acid molecule library was sequenced on a HiSeq 2500 instrument (Illumina), generating between 51 million and 54 million 100 bp pair-end reads per library. The quality-controlled sequence read results were obtained using Tophat (C. Trapnell et al., Bioinformatics, 25, 1105-1111 (2009); D. Kim et al., Genome Biol. 14, (2013).). Ferrophilus IS5 was mapped to the draft genome, giving a minimum mapping identity of 97.4%. To compare the relative expression patterns of the IS5 strain cells cultured in a lactate-starved or lactate-sufficient state, the sequence reading results were quantified using FPKM metrics. FPKM values were further normalized by the average of two housekeeping genes, adenylate kinase (adk) and recombinant protein A (recA).

(Membrane fraction extraction)
Lactic acid starvation (1.58 g) and lactate sufficiency (2.25 g) A 2 L cell culture of ferrophilus IS5 cells was centrifuged at 7197 g at 4 ° C. for 20 minutes to collect a wet pellet. The cells are washed, resuspended in 10 mM Tris-HCl buffer (pH 8) and treated as described in Myers and Myers (CR Myers et al., J. Bacteriol. 174, 3429-3438 (1992)). , EDTA-lysozyme-Brij 58 method. Until a pellet was no longer formed, centrifugation was repeated at 7197 × g at 4 ° C. for 10 minutes to remove debris and undisrupted cells. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 45,900 rpm (RCFmax 177,000 × g) at 4 ° C. for 2 hours using a Hitachi type S50A-2130 rotor to pelletize the membrane mixture. To separate the inner membrane (IM) and outer membrane (OM), the membrane pellet was resuspended in 0.5 ml of 10 mM Na ⁺ -HEPES (pH 7.5) buffer and 35% to 55% prepared in the same buffer. (Wt / wt), and centrifuged at 28,500 rpm (RCFmax 82,000 × g) at 4 ° C. for 17 hours using a Hitachi type S50ST-2069 rotor. IM and OM fractions were separated on different sucrose gradients. The membrane fraction was carefully transferred to a 1.5 ml tube using a pipette. The OM fraction was further purified by washing with 2% Triton X-100 for 10 minutes at room temperature to dissolve the attached IM, and then using a Hitachi type S110AT-2115 rotor at 51,200 rpm (RCFmax 150,000). × g), and centrifuged at 4 ° C. for 2 hours to pelletize. Separation of IM and OM was confirmed by subjecting to sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) to protein staining with GelCode Blue Safe Protein Stain (Thermo Scientific). .

(Spectral analysis of extracted OM fraction)
The protein concentrations in the solution containing the OM fraction extracted from lactate-starved cells and lactate-sufficient cells were 852.1 μg / ml and 5021.0 μg / ml, respectively (about 6 times that of lactate-starved cells). Then, using Shimadzu UV Probe MPC-2200, the OM fractions of lactate-starved cells and lactate-sufficient cells were 0.34 mg (0.4 ml × 852.1 μg / ml) and 2.0 mg (0.4 ml × 5021, respectively). (0.0 μg / ml), and the localization of the c-type cytochrome in the OM fraction was identified (optical path, 2 mm; slit width, 5.0 nm; scan speed, culture medium). Absorption spectra were measured at room temperature under air oxidation and dithionite reduction conditions. For a quantitative comparison of the heme content, the baseline of the absorption spectrum was subtracted and the Soret peak intensity at 419 nm was obtained, normalized to 341 μg of the same protein.

(Identification of gene encoding OM cytochrome)
To identify the gene encoding OM cytochrome, the OM fraction isolated from lactate-starved IS5 cells with high cytochrome content was electrophoresed, and then Thomas et al. (PE Thomas et al., Anal. Biochem. 75, 168-176 (1976)) and heme staining using the tetramethylbenzidine-H ₂ O ₂ method. Five dominant heme-positive bands were excised and digested in the gel overnight at 37 ° C. using trypsin (TPCK treatment, Worthington Biochemical Co.). The digested peptide was analyzed by nano-liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were prepared from 0% to 35% buffer B (100% acetonitrile and 0.1%) using a nano ESI spray column (75 μm [ID] × 100 mm [L], NTCC analysis column C18, 3 μm, Nikkyo Technologies). A linear gradient of formic acid) was separated at a flow rate of 300 nL / min for 10 minutes (Easy nLC; Thermo Fisher Scientific). The mass spectrometer was operated in positive ion mode and MS and MS / MS spectra were acquired using the data-dependent TOP10 method. Predicted in-house using a local MASCOT server (version 2.5, Matrix Sciences; parameters: Peptide Mass Tolerance, ± 15 ppm; fragment mass tolerance, ± 20 mmu; and Max Missed Leaves, 3, equipment type, ESI-TRAP) D. The MS / MS spectrum was searched against the ferrophilus IS5 60229 ORF database (3431 coding sequence in genome and plasmid; 1089117 residues).

(Construction of phylogenetic tree)
D. For the search for homologous proteins to the extracellular cytochrome of ferrophilus IS5, the protein sequence encoded by DFE_450 and DFE_464 of the IS5 strain was used as a query sequence in the NCBI non-redundant database using the blastp algorithm. Proteins with a maximal blast score of 200 or more were considered highly identical. The amino acid sequence of the same protein was collected from the NCBI protein database. For the construction of a 16S rRNA phylogenetic tree, 16S rRNA sequences were collected from the NCBI nucleotide database, aligned using MUSCLE (RC Edgar, Nucleic Acids Res. 32, 1792-1797 (2004)), and MEGA 7 ( Analyzed by the adjacent binding method (N. Saitou et al., Mol. Biol. Evol. 4, 406-425 (1987)) using S. Kumar et al., Mol. Biol. Evol. 33, 1870-1874 (2016). . To construct a protein phylogenetic tree, the amino acid sequence from which the unreliable column was deleted (89.2% of the residues remained) was aligned using GUIDANCE 2 (I. Sela et al., Nucleic Acids Res. 43). , W7-14 (2015)) and MEGA 7 (S. Kumar et al., (2016) supra) using the maximum likelihood method.

(Whole cell electrochemical analysis)
Electrochemical measurements were performed in a three-electrode reactor held in a single COY anaerobic chamber filled with 100% N _2. Indium tin oxide (ITO) (SPD Laboratory, Inc., Japan) grown on a glass substrate by spray pyrolysis was used as a working electrode (resistance 8 Ω / square, thickness 1.1 mm, surface area 3.1 cm ² ). , Placed at the bottom of the reactor. Platinum wire and Ag / AgCl (sat.KCl) were used as counter and reference electrodes, respectively. The salt medium having the following composition was used as an _{electrolyte: 457mM NaCl, 47mM MgCl 2,} 7.0mM KCl, 5.0mM NaHCO 3, 1.0mM CaCl 2, 1.0mM K 2 HPO 4, 1.0mM NH 4 Cl, 25 mM Na ⁺ -HEPES (pH 7.5), 1 mL of tungstic selenite solution (12.5 mM NaOH, 11.4 μM Na ₂ SeO ₃ .5H ₂ O, 12.1 μM Na ₂ WO ₄ .2H ₂₎ O), and 1 ml of a trace element solution SL-10 (described in DSMZ medium 320). In the salt medium, 21 mM Na ₂ SO ₄ as an electron acceptor and 1 mM acetate as a carbon source were added, and no vitamin or reducing reagent was added. The salt medium was sterilized by passing through a 0.22 μm filter and then degassed by purging with 100% N ₂ for 15 minutes. A total of 4.5 ml of sterile oxygen-free salt solution was added as electrolyte to the electrochemical reactor. During the electrochemical measurements, the reactor was operated without stirring. Strain IS5 and D. vacuolatum cells were harvested from 50 ml of culture by centrifugation, resuspended in 0.5 ml of anoxic medium and then added to the reactor to a final OD600nm of 0.1 and 0.3. Chronoamperometry, linear sweep voltammetry (LSV) and differential pulse voltammetry (DPV) were measured using an automatic polarization system (VMP3, Bio Logic Company, France). DPV was measured under the conditions of pulse increment of 5.0 mV, pulse amplitude of 50 mV, pulse width of 300 ms, and pulse period of 5.0 seconds. The LSV was measured at a scanning speed of 0.1 mV / s. The potential scanning was performed in the negative direction.

(Scanning electron microscopy)
After performing the electrochemical measurements, the ITO electrode was removed from the reactor, fixed with 2.5% glutaraldehyde, and then washed by immersing the electrode three times in 50 mM Na ⁺ -HEPES (pH 7.4). The washed samples were dehydrated with a 25%, 50%, 75%, 90% and 100% ethanol gradient in the same buffer, exchanged for t-butanol three times and then lyophilized under vacuum. Dried samples were coated with evaporated platinum and then viewed using a Keyence VE-9800 microscope.

(Transmission electron microscopy)
Lactate starvation and lactate sufficiency in 50 ml of cell culture ferrophilus IS5 cells were harvested by centrifugation at 5000 xg for 8 minutes and immediately fixed on ice in a degassed solution containing 2% paraformaldehyde and 2.5% glutaraldehyde. After fixation, all operations were performed in 2 ml Eppendorf tubes. After gentle resuspension in 1.5 ml of 50 mM Na ⁺ -HEPES (pH 7.4, 35 g / L NaCl), it was washed five times by centrifugation (5000 × g, 4 minutes). According to the method described by McGlynn et al. (SE McGlynn et al., Nature 526, 531-U146 (2015)), cytochrome-reactive 3,3′-diaminobenzidine (DAB) -H ₂ O ₂ staining, OsO ₄ staining, And resin embedding was performed sequentially. To visualize the membrane vesicles, combinatorial heavy metal staining was performed to enhance the membrane contrast. Deerinck et al. (T.J.Deerinck et al., NCMIR methods for 3D EM:. A new protocol for preparation of biological specimens for serial block face scanning electron microscopy Available at https://ncmir.ucsd.edu/sbem-protocol(2010) ) Was modified slightly to perform an initial cell wash using bacterial cells and 50 mM Na ⁺ -HEPES (pH 7.4, 35 g / L NaCl), respectively, instead of mammalian tissue and cacodylate buffer. . The obtained resin block was cut at 80 nm with a diamond knife (DiATOME, Ultra35 °), and the floating section was mounted on a copper microgrid (Nishin EM). Thin sections were examined and imaged using a JEM-1400 microscope operating at 80 kV.

(Fluorescence microscopy)
Lactate-starved D. ferrophilus IS5 cells were fixed with a degassed 2% glutaraldehyde solution, and each was protein-specific as described by Pirbadian et al. (S. Pirbadian et al., Proc. Natl. Acad. Sci. USA 111, 12883-12888 (2014)). And a membrane-specific fluorescent dye, NanoOrange reagent and FM4-64FX (ThermoFisher Scientific). Each fluorescence was observed under a x100 oil immersion objective with a Leica DFC450C epi-fluorescence microscope equipped with a FITC filter and a TXR filter, respectively.

(Example 1) Identification of outer membrane heme protein of S. ferrophilus IS5 The electron uptake process in SRB is based on iron-reducing bacteria (DE Ross et al., PLoSONE 6, e16649 (2011); A. Okamoto et al., Angew. Chem. Int. Ed. 53 Outer membrane (OM) heme protein or nanofilament, as occurs during extracellular electron transfer by LA Shi et al., Env. Microbiol. Rep. 1, 220-227 (2009)). (H. Venzlaff et al., Corros. Sci. 66, 88-96 (2013); D. Enning et al., Appl. Environ. Microb. 80, 1226-1236 (2014)). The present inventors have proposed D.S. The entire 3.7 Mbp circular genome of ferrophilus IS5 was sequenced and 26 genes encoding multiheme cytochromes containing at least four heme binding motifs were identified. They are essential for the formation of cytochrome complexes (OM cytochromes, OMCs) that penetrate the direct electron transport pathway, OM, and for the reduction of extracellular solids in the model iron-reducing bacteria Shewanella oneidenissis and Geobacter sulfurreducens. . G. FIG. The gene encoding the conductive nanofilaments of S. sulfurreducens was not found in the genome of the IS5 strain, but the multi-heme cytochrome encoded by the genes DFE_450 and DFE_464 was predicted to be extracellular, and thus D. It was considered to be a necessary component of extracellular electron uptake in ferrophilus IS5. Searching the NCBI non-redundant protein database, OMCs homologous to those encoded by DFE_450 and DFE_464 reduce SO ₄ ²⁻ , SO ₃ ²⁻ , S ₂ O ₃ ²⁻ , S ⁰ , and Sn ²⁻ The phylum Proteobacteria, Thermodesulfobacteria and Acquifex were widely distributed among cultured and uncultured bacteria of the phylum (Fig. 1a). Notably, D. The homolog of ferrophilus IS5 OMC was obtained from S. cerevisiae. oneidensis, G .; Sulfurreducens and Acidithiobacillus ferroxidans were clearly sequence-different from the OMC (FIG. 1b). Due to the widespread presence of these conserved OMCs in OSS-respiring bacteria, this ill-defined microbial community is able to extract electrons from reduced sulfide minerals via OMC under conditions where soluble electron donors are limited. Was suggested.

Thus, the present inventors performed both OM extraction and transcriptome analysis of cells cultured in a medium having a limited lactic acid content, thereby obtaining a D.D. It was confirmed that ferrophilus IS5 highly expresses OMC. Lactate was completely consumed 5 days after seeding, and the crude membrane fraction was clearly separated into the inner and outer membranes (FIG. 2a). The UV-vis absorption spectrum of the extracted OM fraction showed Soret and Q band absorption characteristic of oxidized and reduced c-type cytochromes (FIG. 2B, left panel). Analysis of the spectral data shows that lactate-starved IS5 cells have at least 10 times more OMC than cells cultured under lactate-sufficient conditions where 30 mM or more of lactate remained after 5 days of culture. (Right figure in FIG. 2B). D. To elucidate the gene encoding ferrophilus IS5 OMCs, the electrophoretic OM fraction of lactate-starved cells was treated with heme staining (3,3 ′, 5,5′-tetramethylbenzidine-H ₂ O ₂ ). OMCs were visualized and the amino acid sequence of the protein in the heme positive band was determined. Using this approach, the gene products of DFE_449 and DFE_461 encoding cytochromes with 12 and 14 heme binding sites, respectively, were detected (FIG. 2a). No other cytochromes were detected in the heme-positive band, but transcriptome analysis showed that other cytochromes, including two extracellular cytochromes encoded by DFE_450 and DFE_464, were overexpressed under lactate-starved conditions (Fig. 2c). This suggested that IS5 cells promoted the expression of OMC for electron uptake when they became starved of electron donors.

The present inventors have found that lactic acid-starved D. It was further confirmed by electrochemical measurements that ferrophilus IS5 cells were able to extract electrons from the electrode surface with significantly higher efficiency than cells with sufficient lactate. Using a three-electrode anaerobic reactor with an indium-tin-doped oxide (ITO) electrode maintained at -0.4 V (compared to a standard hydrogen electrode) to prevent H ₂ generation (X. Deng et al., Electrochemistry). 83, 529-531 (2015)), and the current generated by extracellular electron uptake with sulfate reduction was measured. Lactate-starved D. When ferrophilus IS5 cells were introduced into a reactor where the electrode served as the sole electron donor, the cathodic current density increased immediately and increased to> 0.2 μAcm ⁻² within one hour (FIG. 3a). In contrast, when the SRB Desulfobacterium vacuolatum or sterile media consuming and _{H 2} introduced into the reactor, the increase in current is detected was negligible. Lactate sufficiency D. Addition of ferrophilus IS5 cells did not sharply increase the cathodic current, there was a slight increase in current and a change in background non-Faraday current. As a result, the lactate concentration in the cell culture decreased ferrophilus IS5 cells were shown to alter the electron uptake capacity. The doubling time of IS5 cells is about 13.5 hours under anaerobic growth conditions containing lactate, but after 3 days the number of lactate-starved IS5 cells on the ITO electrode has not increased significantly, while the cathodic current of IS5 cells has not increased. Generation continued. This suggests that the electrode provides much less energy to IS5 cells than lactate. The potential dependence of the sulphate reduction current measured by linear sweep (LS) voltammetry after cathodic current measurement indicates that sulphate reduction is initiated at -0.3 V in lactate-starved IS5 cells and reaches a maximum at a potential of -0.42 V. (Fig. 3b). In contrast, lactate sufficiency IS5, _{H 2} consumption D. No significant current for sulfate reduction was observed by LS voltammetry of vacuolatum or sterile medium. The observed onset potential for sulfate reduction of -0.3 V was determined by differential pulse voltammetry, which was determined by D.C. This was consistent with the redox profile of ferrophilus IS5.

In particular, another peak observed in LS voltammetry of lactate-starved cells at -0.1 V can be assigned to the reduction of biosynthetic iron sulfide, which is the peak current after 2 hours in lactate-starved IS5 cells. The reason for the slight decrease in current after generation is explained by the fact that the production of iron sulfide can inhibit direct contact between cells and the electrode surface (FIG. 3A). The threshold electrode potential for -0.3 V sulfate reduction is similar to the redox potential of NADH, but more positive than ferredoxin, which indicates that NADH transfers electrons from periplasmic cytochrome to cytoplasmic adenosine phosphosulfate reductase. It suggests that it serves as a receiving primary electron transport component (FIG. 3c). These results further suggest that the identified electron uptake mechanism requires almost a minimum amount of energy in the form of electrons to drive the sulfate reduction process. The formation of the most important agents (for example, menaquinone) that donate electrons to other OSSs (SO ₃ ²⁻ , S ₂ O ₃ ²⁻ , S ⁰ and Sn ²⁻ ) also has a positive oxidation equivalent to or higher than that of NADH. As they occur at the reduction potential, the new group of OMCs identified here can be an important component of the microbial energy acquisition process of OSS respiring bacteria, especially in environments confined to electron donors, including marine sediments. In addition, the growth of IS5 associated with electron uptake was negligible on the electrodes, but the minimal but sufficient energy requirements to perform metabolism was very high, as was observed in the underground poor environment. It may be associated with a slow growth rate (TM Hoehler et al., Nat. Rev. Microbiol. 11, 83-94 (2013)).

(4) Lactic acid-starved IS5 cells formed a monolayer biofilm on the electrode by scanning electron microscopy observation of the electrode surface after the electrochemical measurement (FIG. 4a). This is similar to many bacterial strains with extracellular electron transfer capacity, consistent with the concept that electron uptake occurs via OMCs. Furthermore, nanofilament structures were observed to extend between cells and from cells to the electrode surface (FIG. 4b). S. Similar nanowires produced by oneidensis MR-1 are available from OM (S. Pirbadian et al., Proc. Natl. Acad. Sci. USA 111, 12883-12888 (2014); YA Gorby et al., Proc. Natl. Acad. Sci. USA 103, 11358-11363 (2006)), it is considered that cytochromes detected in the OM fraction of the IS5 strain are also localized in the observed nanowires.

In response to lactate restriction, as observed for OMC, The formation of nanowire-like structures was induced in ferrophilus IS5 cells. In order to examine the distribution of OMC on the nanowire, transmission electron microscopy was performed on thin sections of cytochrome-reactive 3,3'-diaminobenzidine (DAB) -H ₂ O ₂ stained cells. Center catalyzed the formation of DAB polymer while exhibiting high binding affinity for OsO ₄ . As a positive staining control, S. cerevisiae grown under conditions that promoted nanowire formation. oneidensis and G.W. sulfurreducens cells were used (YA Gorby et al., Proc. Natl. Acad. Sci. USA 103, 11358-11363 (2006); G. Reguera et al., Nature 435, 1098-1101 (2005)). Consistent with the expression analysis of OMCs, the outer edges of lactate-starved IS5 cells (FIGS. 4c-e) stained at approximately twice the intensity of lactate-sufficient cells. S. oneidensis and G.W. A similar staining intensity was observed in S. sulfurreducens cells, whereas E. coli cells without OMC used as a negative staining control showed stronger staining in the cells compared to the membrane area. D. The ferrofilus IS5 nanowire resembles a bamboo-like structure with a diameter of 30-50 nm and is clearly visible only with positive DAB staining, suggesting a high cytochrome coverage on the nanowire surface. IS5 nanowires are available from slightly thinner than that of S. oneidensis, but both show the same segment structure and strong cytochrome positive staining, and the IS5 nanowires also have It has been suggested to play a role in electron transfer as proposed in oneidensis. In contrast, G.A. sulfurreducens nanowires had a diameter of about 7 nm and exhibited both weakly positive and negative DAB staining. These differences are described by S.M. oneidensis nanowires are consistent with previous structural models, suggesting that they are an extension of the OM (S. Pirbadian et al., Proc. Natl. Acad. Sci. USA 111, 12883-12888 (2014)). . sulfurreducens mainly consist of type IV pilin PilA (G. Reguera et al., Nature 435, 1098-1101 (2005)).

D. To examine the composition of the ferrophilus IS5 nanowires in more detail, lactate-starved cells were stained with protein-specific and membrane-specific fluorescent dyes, NanoOrange and FM4-64FX, respectively (Figures 4f and g). . Fluorescence microscopy of the IS5 strain nanowire stained for both protein and membrane showed that the nanowire was an extension of the cell membrane and that oneidensis nanowires (S. Pirbadian et al., (2014) supra). Together with the bamboo-like structure observed with a transmission electron microscope, the strain IS5 and S. The oneidensis nanowire was considered to be an ordered alignment of OM vesicles of several tens of nanometers in size. Heavy metal staining was performed using a transmission electron microscope to emphasize the contrast of the membrane, and the secretion of membrane vesicles and their alignment in lactate-starved IS5 cells were observed. Thus, it was shown that the extracted OM fraction of the lactate-starved IS5 cells also likely contained nanowires. These data are available from It is suggested that OMCs and nanowires of ferrophilus IS5 mediate electron uptake from extracellular solids.

As identified in several iron-reducing bacterial strains, the identified multihemocytochromes are It was thought that a protein complex could be formed through the OM of ferrophilus IS5. The cytochromes encoded by DFE_449 and DFE_461 detected in the extracted OM fraction are predicted to be in the periplasm, and these genes are located in the genome close to DFE_450 and DFE_464, which are predicted to encode extracellular cytochromes Therefore, DFE_449 and DFE_461 are periplasmic decahemocytochromes in the OM MtrCAB cytochrome complex and are important for metal reduction of Schwanella species (AS Beliaev et al., Mol. Microbiol. 39, 722-730 (2001); C. Reyes et al., J. Bacteriol. 194, 5840-5847 (2012).); It was thought to encode an OM-bound periplasmic cytochrome that functions similarly to oneidensis MtrA. From transcriptome analysis, as seen for β-barrel proteins in iron-reducing bacteria (RS Harthorne et al., Proc. Natl. Acad. Sci. USA 106, 22169-22174 (2009)), protein-protein interactions Equivalent expression levels of genes DFE_448-451 and DFE_461-465, which encode proteins containing three other cytochromes and two NHL-repeat β-propeller proteins that can function as sites and stabilize OMC, are apparent. (FIG. 2c). Homologous β-propeller proteins have also been identified in a number of OSS-respiring bacteria in marine sediments, suggesting that potential OMC complexes may be widely conserved among this microbial community. Similar electron uptake has a different OMC (Cyc2) than IS5 (FIG. 1b) (T. Ishii et al., Front. Microbiol. 6, (2015); A. Yarzabal et al., J. Bacteriol. 184, 1502-1502) (2002)) Fe ²⁺ oxidation Ferrooxidans also reported that bacterial oxidation of sulfide minerals under anaerobic conditions is considered to be indirect oxidation of Fe ²⁺ ions released from iron sulfide (KL Straub et al., Appl. Environ.Microb.62, 1458-1460 (1996); CJ Jorgensen et al., Environ.Sci.Technol.43,4851-4857 (2009)), which is different from the direct electron uptake model shown in the present invention.

に お い て In various uncultured SRB strains mixed and cultured with methane archaea, As the genes encoding OMCs that perform anaerobic oxidation of methane homologous to the OMCs of ferrophilusΔIS5 have been identified (FIG. 1), various genes that form consortia with methane-utilizing archaea (FIG. 1) Once identified, the observed D. The electron uptake capacity of ferrophilus {IS5} OMCs supports a recently proposed model in which SRBs can accept electrons directly from archaeal cells via OMCs or nanowires (SE McGlynn et al., (2015) S. Scheller et al., Science 351, 703-707 (2016); G. Wegener et al., Nature 526, 587-U315 (2015)). According to the electrochemical data, the SRB in the methane oxidation consortium requires electrons with a negative potential less than -0.3 V, which is due to the electrons from methane-trophic archaea in 2,7-AQDS (- (Redox potential of 0.185 V) (S. Scheller et al., (2016) supra). These findings, which relate to the energetics of identified clade OMCs and electron transfer processes mediated by these heme proteins, suggest that microbial energy production of OSS-induced microorganisms in sediments under limited conditions of soluble energy sources In addition, it is expected to deepen the understanding of interspecies electron transport in synthetic consortiums containing SRB.

Since the protein of the present invention is involved in electron extraction from the outside of the cell, by inhibiting the expression or function of the protein of the present invention, it is possible to inhibit events based on electron extraction in bacteria, and Corrosion and the like can be prevented or prevented.

Claims (23)

1. An amino acid sequence according to one sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18; SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and An amino acid sequence having 80% or more identity to the amino acid sequence described in one sequence selected from SEQ ID NO: 18; one amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 An amino acid sequence in which 1 to 20 amino acids have been substituted, deleted, inserted or added in the amino acid sequence described in the sequence; or SEQ ID NOs: 2, 4, 6, 8, 10, 12, 12 A cell surface protein having an amino acid sequence having a homology score of 200 or more with the amino acid sequence described in one sequence selected from 14, 16, and 18.
2. タ ン パ ク 質 The protein according to claim 1, which has an extracellular electron transfer effect.
3. The protein according to claim 1 or 2, which is a cytochrome enzyme or a β-propeller protein.
4. [4] A nucleic acid molecule having a base sequence encoding the protein according to any one of [1] to [3].
5. A base sequence described in one sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 9, 11, 13, 15 and 17, or SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, The nucleic acid molecule according to claim 4, which has a base sequence that binds under stringent conditions to a nucleic acid having a sequence complementary to the base sequence described in one of the sequences selected from 15 and 17.
6. (4) A vector comprising the nucleic acid molecule according to claim 4 or 5.
7. (7) A host cell having the vector according to (6).
8. The method according to any one of claims 1 to 3, which comprises culturing the host cell according to claim 7 to express the protein according to any one of claims 1 to 3. A method for producing a protein.
9. An antibody, an immunoreactive fragment thereof, or an aptamer that binds to the protein according to any one of claims 1 to 3, or an antisense that binds to the nucleic acid molecule according to claim 4 or 5. A nucleic acid, dsRNA, or aptamer.
10. The antibody or the immunoreactive fragment thereof, the aptamer, the antisense nucleic acid, or the antibody according to claim 9, which has an activity of inhibiting the extracellular electron transfer activity of the protein according to any one of claims 1 to 3. dsRNA.
11. A composition comprising the antibody according to claim 10, or an immunoreactive fragment thereof, an aptamer, an antisense nucleic acid, or dsRNA.
12. (4) A method for determining a protein that causes corrosion of a metal material, including identifying an electron transfer enzyme from cell surface proteins of a bacterium present on the surface of the metal material.
13. The method according to claim 12, wherein the identification of the electron transfer enzyme is performed by determining a protein having an electron transfer reaction center.
14. An amino acid sequence having at least 80% identity to the amino acid sequence described in one of the sequences selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; The method according to claim 12, which is performed by selecting a protein having the following.
15. An antibody that binds to an electron transfer enzyme determined as a causative protein of a metal material by the method of claims 12 to 14, or an immunoreactive fragment or aptamer thereof, or an antisense nucleic acid that binds to a gene encoding the electron transfer enzyme; A method for producing a corrosion inhibitor, comprising producing dsRNA or an aptamer.
16. An electron transferase is identified from a cell surface protein of a bacterium present on the surface of a metal material by the method according to claims 12 to 14, and the determined antibody that binds to the electron transferase, an immunoreactive fragment or aptamer thereof, or A method for producing a corrosion inhibitor, comprising producing an antisense nucleic acid, dsRNA, or aptamer that binds to a gene encoding the electron transferase.
17. (4) A method for preventing corrosion of a metal material, which comprises inhibiting the function or expression of the electron transfer enzyme in bacteria existing on the surface of the metal material.
18. 方法 The method according to claim 17, wherein the electron transfer enzyme is the protein according to any one of claims 1 to 3.
19. {When the function or expression of the electron transfer enzyme is inhibited, the potential of the metal material having a negative potential is set to −0.32 V vs. 18. The method of claim 17, wherein the method is performed by stopping the electron withdrawing metabolism of the bacteria above SHE.
20. 19. A method according to claim 18, comprising contacting the composition according to claim 11 with a surface of a metallic material.
21. 18. The method according to claim 17, comprising contacting the antibody or aptamer produced by the production method according to claim 15 or 16 with a surface of a metal material.
22. Identification of electron transfer enzymes from bacteria present on the surface of metallic materials; and
    A method for preventing corrosion of a metal material, comprising inhibiting the function or expression of an electron transfer enzyme in bacteria existing on the surface of the identified metal material.
23. A method for preventing corrosion of metal materials installed in an external environment,
    Collecting a sample containing bacteria having cell surface enzymes from the external environment;
    Identifying a cell surface electron transfer enzyme present in the sample;
    Preparing an antibody that binds to the cell surface electron transferase or an immunoreactive fragment or aptamer thereof, or an antisense nucleic acid, dsRNA, or aptamer that binds to a gene encoding the cell surface electron transferase;
    A method for preventing corrosion of a metal material, comprising flowing the antibody, aptamer, antisense nucleic acid, or dsRNA through a pipe.

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