JPH07501201A - Ammonia production by recombinant nitrogen-fixing bacteria - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 4. As of August 30, 1991, the National Co., Ltd. ion of Industrial and Marine Bacteri a Ltd,, Aberdeen, 5cotland, UK accession number 4 0438, or its strain with the same ammonia overproduction function. 4. The bacterium according to claim 3, which is a mutant or variant.

5. Any one of claims 1 to 4, wherein the mutant bacterium-like gene according to one of claims 1 to 4 is Steps to isolate the negative genes of the parent nitrogen-fixing bacteria that are regulated only by similar genes. a step of mutating the nif^ or The four genes of the parent nitrogen-fixing bacterium were mutated while preserving the functionality of the similar genes. and replacing it with a n1fL or recirculation-like gene.

6. Transferring the concave gene from one bacterium (recipe end) to another bacterium (donor) 6. The method according to claim 5, wherein the mutant niflyu gene is substituted.

7. Ensure that the donor and recipient end bacteria are of the same genus or family. The method described in claim 6.

8. The method according to claim 7, wherein the donor and recipient bacteria are of the same species. Law.

9. Claim 7, wherein the donor and recipient bacteria are Zotobacter. The method described in.

10. The donor and recipient bacteria are Zotobacter vinyl 9. The method according to claim 8, wherein the andii.

11. The donor and recipient bacteria are the National Co11ection of Industrial a nd Marine Bacteria Ltd, Aberdeen, 5 cotland, the strain deposited in the UK under accession number 40438, or the same strain. A claim that the invention is a mutant or variant thereof that has growth and ammonia production functions. 8. The method described in 8.

12. A method of providing a fixed nitrogen source to plants, comprising any one of claims 1 to 4. or according to any one of claims 5 to 11. A method consisting of introducing mutant nitrogen-fixing bacteria into nitrogen-fixing symbiosis with plant tissues. .

13. The method according to claim 12, wherein the mutant bacterium is introduced into the soil before or after planting. How to put it on.

14. According to claim 12, the mutant bacterium is introduced into the plant before or after planting. Method described.

15. The method according to claim 12, wherein the mutant bacterium is introduced into seeds before or after planting. How to put it on.

16. The method of claim 12, wherein the bacteria are introduced as an aerosol or liquid. .

17. The bacteria have the ability to bind to lectins produced by legumes. 13. The method according to claim 12.

The method according to claim 18, obtained from Ieguminosarum.

20. A fixed nitrogen source is provided by the method according to any one of claims 12 to 19. The plants that were harvested.

21. Mutation site for insertion into the recipe end bacterium by gene replacement Denji.

22. As of August 30, 1991, the National Co., Ltd. ion of industrial and Marine Bacteria Ltd,, ^berdeen, 5cotland, UK accession number 4043 A, vinelandii strain deposited in 8, or the same growth and ammonia strain characterized by being derived from a mutant or variant thereof that has a near-producing function The gene according to claim 21.

Specification Ammonia production by recombinant nitrogen-fixing bacteria Field of invention The present invention is in the field of ammonia production by nitrogen-fixing bacteria.

Conventional technology Numerous microorganisms contain the major bioelements in inorganic form (i.e., carbon, nitrogen, sulfur, hydrogen, and can assimilate oxygen and oxygen). The ability to use NZ as a nitrogen source is limited to prokaryotes. Among them, it is relatively rare. Enzyme system responsible for N2 fixation is called nitrogenase. Biosynthesis of nitrogenase system components is 15-2 Determined by 0 types of box genes.

Free-living nitrogen-fixing bacteria fix enough atmospheric nitrogen to meet their own needs. the There is evidence that nitrogen-fixing bacteria have a significant Large amounts of ammonia are rarely observed. Box gene transcription and/or nitrogen Regulatory mechanisms that control enzyme activity meet the growth and survival needs of bacterial cells. Cellular energy is wasted by fixing more nitrogen than is needed to Guaranteed not to happen. Particularly in excess of environmental ammonia or oxygen, free Prevents the expression of the box gene in active diazotrophs (nitrogen-fixing bacteria) . Ammonia renders nitrogenase useless, and oxygen deactivates the enzyme. mosquito Bacteria produce assimilated ammonia in the form of ammonium cations (NH4+). However, the term "ammonium" will be used hereafter for brevity.

Attempts to induce ammonium secretion have so far been limited to ammonium The focus is on physiological suppression or genetic manipulation of enzymes involved in anabolism. Blue-green bacteria Inhibition of glutamine synthetase (GS) The substance is treated with monomethionine-DL-sulfoximine (MSX). and mutants with 0.3-7mM NH,+ in the growth medium, up to 1,6 secreted up to mM NH4'' (Polr et al., 1986. J, Ba cteriol,, 165.412-419. and Th-3504). True detail Among bacteria, Gln- or Klebsiella pneumoniae GlrVasm-1 mutant (Anderson et. 5, 1977, J, Gen, Microbiol,, 103. 107-122 and Shanm Ugam et al., 1975.　Proc, Natl, ^cad, Sc i, USA, 72. 136-139), Rhadob with modified GS production acter capsulatus and zospirillum brasi mutants of Lense (Wall et al.

It has been reported that ammonium secretion occurred. Significant ammonium secretion was observed. However, these chemically treated or mutated microorganisms require a large amount of access to growth. Requires supplementation of amino acid glutamine.

The regulatory pathways and mechanisms involved in repression of digenes by ammonium are Diazotrophs Klebsiella pneuIIloniae and and Zotobacter vineLandii. ing. The main points of interest in both microorganisms (and other Gram-negative nitrogen-fixing bacteria) are The transcription of other citrate genes required for the assembly and activity of nitrogenase should be activated (actively However, these The two genes are expressed simultaneously. NIFL protein contains ammonium Then, even if it is at a relatively low level (>about 5 μM), it will lead to NIF^. together to inactivate it. Higher ammonium levels (>about 20+?Ctan) This protein is required in cells grown with ammonium. The protein is dephosphorylated and therefore inactive. That is, pneus+o Inhibition of nitrogenase synthesis by ammonium in A. niae There are two mechanisms: inactivation of NIF^ and interference with expression by dephosphorylated NTRC. Happens at the bell.

^, Nitrogen fixation in P. vinelandii is biochemically and genetically distinct. determined by the presence of three nitrogenase enzymes, each of which has a are synthesized under different metal supply conditions. ferments purified from numerous other nitrogen-fixing bacteria. Molybdenum nitrogenase, which is similar to NT, differs in its construction and activity. RC is not required for nitrogen fixation, which suggests that the partial reading frame (0RF ) represents (1le1nettet al,, 19861Mo1. Microbiol, 2.315-321).

Ammonium is produced by the nitrogen-fixing bacteria mentioned above for various purposes, and this means that , meaning that nitrogenase synthesis does not take place and the microorganisms are unable to fix nitrogen at all. do.

The problem is that nitrogen-fixing bacteria can be removed from ammonia by deregulating nitrogenase production. Attempts to produce ammonium assimilate enzymes were unsuccessful, and genetically engineered ammonium assimilation enzymes Although attempts to produce ammonia by cultivating ammonia have been successful, The microorganism itself cannot reproduce normally.

It was found that mutant bacterial strains were produced that produced and secreted ammonium in significant amounts. I made it clear. Even more surprisingly, such mutants grow similarly to the parent strain.

Alternatively, the n1fA-like gene may further contribute to the construction of nitrogenase and the mutation of active nitrogen-fixing bacteria. Variants are provided.

Preferred bacteria are those present in the rhizosphere or attached to or on the roots of plants. Inside the trunk (so-called systemtsic endophytes) Those that usually live in strong symbiosis with plants by living in All bacteria from the genus Azotobacter, whether or not. Special Preferred bacteria include Azotobacter genus, especially Zotobacterium It is derived from R. vinelandii. The most preferred strain is Budapest As of August 30, 1991, 23 St., Machen Dr. ive, ^berdeen, AB21RY, 5cotland, Unit The National Co11ection in ed Kingdom of Industrial and Martine Bacteria L td, (NCIMB) and has been given accession number 40438. The subject of the patent deposit or a mutant thereof having the desired growth and ammonia production functions It is a variant or variant.

According to a second aspect of the invention, e.g. but (if it is downstream) interferes with the expression of the downstream nif^ gene. It is necessary not to do so. In the presence of high levels of ammonium in the culture medium, It will be clear to those skilled in the art that variations can be made in a variety of ways. Mutations are deletions and by combining insertions into other mutants”) or rendering the protein non-functional. By replacing the nucleic acid sequence with one that makes the protein functional, the gene can be A single base exchange in the coded DNA may be sufficient. Those who carry out mutation It is clear to those skilled in the art that the method typically involves one operator under the control of one promoter. competency needs to be conserved in the mutant. In other words, the nif^ gene is the parent It needs to be preserved in the same way as in living organisms, but if it has been modified, the changes must be Must be non-disturbing. For example, a point mutation introducing a termination signal into n1fL nif^ production by mutation, either by insertion or by introducing polar mutations. It is important that life is not interrupted. That is, mutations do not interfere with mRN^ production. It needs to be something good.

It is known that it is similar to For example, to generate a strong promoter Mutations that result in nif should not result in the replacement of promoters with stronger promoters. n1fLA The operon's promoter is stronger than the natural promoter found in wild-type bacteria. The natural promoter is preferred.

The present invention is carried out in the deposited strain Azotobacter vinelandii. I own one tar.

According to a fourth aspect of the invention, the level of ammonium production by the above-mentioned bacteria is controlled. A method is provided. High pH inactivates nitrogenase, so ammonia ammonium secreted by the increase in pH of the medium that accompanies ammonium secretion. It is known that the amount of cells with calcium alginate particles or vinelandii or other nitrogen fixers as mentioned above. Buffering of the medium when producing ammonium using the n1fL mutant of the microorganism p by either increasing ammonium capacity or removing it when it is secreted. H and can raise or lower ammonium production levels as needed. .

The present invention is directed to the use of lectins that are normally abundant in the rhizosphere or in plant roots. It binds to glycoproteins and actually lives in the roots and trunks of plants (systemic endophytes). It is particularly effective against nitrogen-fixing bacteria that are known to coexist with certain plants by .

According to the fifth aspect of the present invention, the mutant nitrogen-fixing bacteria as described above (in plant parts) by leading to a nitrogen-fixing symbiosis with plant tissues (including seeds, whole plants, etc.). A method of providing a fixed nitrogen source to plants is provided. There are several nitrogen-fixing symbiotic relationships with plants. It can be manifested in the following ways. Once bacteria enters a nitrogen-fixing symbiosis with plants, they reside in the rhizosphere. or coexist with plants as described above. Mutant nitrogen-fixing bacteria in plant tissue can be introduced in a variety of ways. They can be applied to the plant itself before or after planting. It can be applied to the soil before or after or to the seeds. Bacteria as an aerosol Alternatively, it is convenient to apply in liquid or solid form.

The mutant bacteria are completely different from the wild-type bacteria normally found in nitrogen-fixing symbiotic relationships with plants. They may be of different families, genera or species.

Mutations in the n1fL gene can be made by mutating wild-type bacteria themselves by the method described above. It can be introduced by mutating or by gene replacement. You can also do that.

The bacterium may be a mutant of a wild-type bacterium that is normally found to coexist with plants. genetics Child replacement involves replacing a gene from one microorganism with a sudden replacement, in this case from another microorganism. This includes replacing the mutated n1fL gene. Two microorganisms are in the same family - or genus, and more preferably from the same species or strain. Yes.

The present invention furthermore introduces a fixed nitrogen source into plants that normally lack symbiotic nitrogen-fixing bacteria. A method is also provided. Mutant bacteria that provide an ammonium source to such plants are Zotobacter vinelandii that exists in the roots of plants is B15hop et al., 1977, describe the construction of Sci. Lectin binding ability can bind to plant roots or other tissues that carry genes that are typed to give can. A. vinelandii n1fL mutant plant roots or other By binding to tissues, fixed nitrogen can be supplied directly from bacteria to plants.

Figure 1 shows the ^zotobacter vinelandii n1fL^ operon. The restriction map and plasmid construction of.

Hereinafter, embodiments of the present invention will be described by way of examples.

The cells were grown under aerobic conditions at 30° C. in culture medium. 2 in a 125m1 flask 5n+1 cultures were incubated on a rotary shaker (180 rpm). con Pittens medium (competence 5 diun, CM) contains Fe and Mo salts. Also in Burk's sucrose medium prepared without the addition of. E, increase colt LB medium was used for growth. In plasmids or genomic transformants Antibiotics with previously unreported subsequences to select for resistance genes Approximately 300 bp of the 3' end of the protein-encoding gene was inserted into the plasmid pDBL5 ( Bennett et al., 1988. Mo1. Microbiol, 2. 315-321. upper reaches To isolate and clone the entire gene, the vinelandii genome Plaques derived from the λ library of DN^ were labeled with 3”P-dCTP DNA. Screened for hybridization with the probe. This probe has 1 Reference) (Bennett et at, 1988. Mo1. Micr obiol, 2゜315-321). Descendants of one hybridized plaque In the insert DNA prepared by Rikita et al., an approximately 12.5 kb EcoRI fragment was found. was identified and subcloned into pBR325 to obtain pAB21. . The size of this 12.5 kb fragment is such that it does not hybridize to the n1fA probe. It contained a genome previously reported to have been isolated (Santero et al. ,, 1988 Mo1. Microbiol, 2゜303-814). p From the limiter knob on the AB21 insert, it is reported by 12.5kb (see above). fields for restriction sites corresponding to those reported, as well as any subclients derived for this task. The loan is shown in Figure 1.

Methods for plotting and screening lambda libraries and DNA flags The method for cloning the 89. Mo1ecular Cloning, Co1d SpringHa rbor Laboratory Press, Co1d Spring H standard methods as described in arbor, and as provided by the restriction enzyme supplier. This was due to the usage procedure.

Example 3: Transformation of A. vinelandif as previously described ( Page et al., 1978. Can, J.

MicrobioL, 241590-1594). Competent cells were generated. in liquid competence medium (CM) before transformation. Instead of growing the cells on CM agar, immediately after the second growth on CM agar, Cells were placed in 1 L of liquid CM + 16 mM Mg5O at a concentration of approximately 10' cells/a + L. Resuspend with a bow. Approximately 50 μm of resuspended cells were dropped onto a 0M agar plate and ．． 1-1 μg of plasmid DNA was mixed with the cells. Incubate for 2 days at 30℃ After incubation, transfer approximately 5 x 10' cells to selective medium containing appropriate antibiotics. did. Cells not mixed with DNA were plated as controls. The transformant is 1 04-10S (μg DNA) −1.

The KIXX cassette containing h) was transformed into plasmid pUC4-KIXX (Phar Plant termination was performed using mase and deoxynucleotides (Figure 1). KIX The resulting plasmids pAB29 and pA have the X cassette inserted in opposite directions. B30 as described in Example 4, two types of vinelandii strains, viz. Transformed into wild type UW136 and the concave-box fusion strain MVIOI. Km’transformation conversion to carbenicillin (CBr1)61icillin) resistance (Cb'). and screened. CB” derivatives contain KIXX-containing wild-type 4/concave-like genes. It is presumed to have arisen from a double-crossover recombination event in which the DNA is replaced by It will be done. The Km'CB' transformant of υ1136 was. pAB29 derivative strain MV3 76 was Nif''''. MY376, MV378 and M digested with 5alI V380-derived (7) DNA (7) Southern blot shows 32P derived from the mutation region. Labeled probe (0.3kb 5atI-3phI fragment derived from pAB31 (see Figure 1).

Southern hybridization experiments showed that in all three mutants, 3. The 5 kb wild-type 5alI fragment is absent and the 6.2 kb hybrid It turns out that it has been replaced by is fragment.

Insert KIXX into the four genes of vinelandii in either direction. also results in the Nff' phenotype, but T during A, vinelandii ni Insertion of n5 or Ω (transcription terminator) cassette results in N1f-phenotype. It was. Therefore, the n1fLA gene is probably found in the throat during K. pneumoniae. It is also transcribed at the same time in Vinelandii. upstream to interfere with transcription. The KIXX aph cartridge inserted into A, vinelandii The n1fL (KIXX) mutation can inhibit the expression of downstream genes. The n1fA gene is clearly expressed in MV376 and MV378.

This may be due to unexpected promoter activity within the cassette or Kt within the n1fL region. It may have arisen from the promoter uniform sequence generated by the XX insertion.

When inserting IXX (MV380) , the level of β-galactosidase assembly was lower than when inserted in the opposite direction (MV378). The price has increased somewhat. However, too many TPs interfere with nitrogenase synthesis and activity. Or NU.

4 Because it turns to toxicity, nif+^, vinelandii background Possibly fatal in most cases.

Example 5: Enzyme assay Nitrogenase and β-galactosidase activities were measured as previously described. (Sambrook et al, 1989. Molecular Cl oning”, Co1d Spring Harbor Laboratory Press.

Col1d Spring) labor, and llalmsley et a l,, 1991. ^ppl.

Environ, 1crobio1. ．． 57. 522-524).

The results of this assay are shown in Table 1 below.

Table 1 UW136 wild type 48° O MV376 n1fL1:KIXX 48 46MVIOI n1fL1:1a cZ 15841b432MV378 n1fH1:1acZ 11967 3 772-N N-free medium.

a Nitrogenase activity in U11L36 and MV376: acetylene reduction (Ethylene produced per minute per 111 mg of protein) (n■ol)).

b MVIOI, MV378. MV3801Nio + t6 B-Caracto fern activity (measured in Miller units (26)).

6 values are the average of 2-3 independent measurements. Deviation from the average is less than 10% Met.

From the data in Table 1, MV376 is Nif+ and N-free medium and 15mM Ammonia (concentration that suppressed nitrogenase activity in wild type strain 01136) It showed wild-type acetylene-reducing activity in both media containing Ni. Similarly, MV3 Both 78 and MV380 react with β-galactosylate in the absence or presence of ammonium. However, in the strain MVIOI, 15aM ammonium Therefore, activity was suppressed.

^, Other activities suppressed by ammonium in vinelandii is the activity of nitrate reductase and nitrite reductase (Luque et al. al., 1987. Arch, Microbiol, 148°231 -235), their expression requires the products of both ntrC and rpoN genes. (Luque et al, supra, 5antero et al.

The difference did not affect normal nitrate reductase regulation.

This activity was observed in NO,-proliferative cultures of wild-type [IW136 and mutant IJV376]. N)J4” (+N0x-) was present in the growth culture solution. (data not shown).

The above results show that in A, vfnelandH, the gene upstream of n ^ is ammonia. mediates the suppression of nif gene expression by ammonium, but the small amount under ammonium control It is shown that the expression of at least one other gene is not mediated. This phenotype is , n1fL is located immediately upstream of 4, X, pneumoniae mutation somewhat similar to K. pneutaoniae n1fL mutant Also, in the S. vinelandii n1fL mutant, nitrogenase synthesis was growth is far more immune to ammonium suppression. ^.

The translation of the entire upstream gene sequence (approximately 1.4 kb) of vinelandji is NTRB rather than the protein with high homology to Pneumoniae n1fL. (same part 24% vs. 31%).

Example 6; Ammonium capacity vinelandii wild-type strain UW136 and mutant strains grown on nitrogen. Culture supernatants with MV376 were tested for the presence of ammonium.

Culture fluid samples were taken at various time points and centrifuged. A in 0.5 ml of supernatant or filtrate. The presence of ammonium was determined using the indophenol method (Bergersen, 1980. "Methods for evaluating biol.

"gical nitrogen fixation", John 1file Y & 5ons Ltd., London). This is the order a) 0.5ml phenol-sodium nitroprusside solution (phenol 50g-/-' + sodium nitroprusside 0.25g-1-'); b) 0 ．． 5+al of sodium hypochlorite solution (0.1 m); and c) 2 m+1 of evaporated It consisted of adding distilled water. The mixture was incubated for 30 minutes at room temperature. 6 The absorption at 25 nm was measured and the ammonium concentration was determined using the same reagent solution. It was estimated from a standard curve obtained using ammonium solutions with various concentrations determined.

The results are shown in Figure 2. Here, * indicates UW136, and mouth indicates MV376.

In contrast to 01136, MV376 grows quite rapidly toward the end of the exponential growth phase. secreted ammonium. Final amount measured in 1IV376 stationary phase culture was 5-10+aM. Half of the supplied sucrose remained during the resting period. Therefore, secretion was not limited by carbon source availability.

Example 7: Construction of n1fL deletion mutant of ^vinelandii Carrying the concave region plasmid pAB27 was digested with BglII, and pAB27, which is responsible for gene cartridge engineering, was digested with BglII. 3.8 kb BamHI fragment derived from J017 (Hynes et al. , Gene, 198978.111-120). saq gene confers sucrose sensitivity upon transfer to certain Gram-negative bacteria. In other words, ± The microorganisms that carry the gene cannot grow in sucrose-containing media. obtained plus Mid pLA5: Sac-Km is responsible for the seat cartridge, in this case ± The cartridge has been replaced with a 585 base pair BglII fragment, The 5' end of the gene and 20 base pairs upstream of the translation initiation codon are deleted. pLA 5:5ac4 tatemycin resistant colonies were selected. These are sucrose-containing media. Although it cannot grow on land, it can grow normally on glucose as a carbon source. It turned out that. Purification of a single colony several times results in stable sucrose-sensitive, bacterial It had a namycin resistance phenotype. This mutant is 1lV399, N1 had an f-phenotype.

Concave: To replace a locus mutation with a deletion mutation that is not mediated by ±DNA. For this purpose, pAB27 was digested with BglII and ligated to itself. Plasmid pLA 4 contains a 585 base pair BglII deletion within the n1fL gene. It turns out. MV399 was transformed using pLA4, and sucrose-containing ammonia Colonies that grew in um-free medium were selected. These Nif+ colonies is also sensitive to kanamycin, which indicates that the sac-Krm cartridge Indicates that it has been replaced.

One such colony was purified and tested for ammonium secretion. this Strain 11V44Q secreted up to 10 aM ammonium into the growth medium. Understand, the pHct of the culture solution when no more ammonium is secreted is approximately 8. ．． 5 Draw each frame.

time (hours) Submission of copy of amendment (Article 184-8 of the Patent Law) Watari Aso, Commissioner of the Patent Office March 8, 1994, 11, Patent Application Indication PCT/GB 9210036 82. Title of the invention Ammonia production by recombinant nitrogen-fixing bacteria 3. Patent applicant Address: United Kingdom, London, Nisui@I.6.φB. Nisui, Nisui With tonton causeway 01 Name: British Technology Group Limited 4. Management: Person: Yamada Building 6, 1-1-14 Shinjuku, Shinjuku-ku, Tokyo, list of attached documents (1) One translation of the written amendment 12. A method of providing a fixed nitrogen source to plants, comprising any one of claims 1 to 4. or according to any one of claims 5 to 11. A method consisting of introducing mutant nitrogen-fixing bacteria into nitrogen-fixing symbiosis with plant tissues. .

13. The method according to claim 12, wherein the mutant bacterium is introduced into the soil before or after planting. How to put it on.

14. According to claim 12, the mutant bacterium is introduced into the plant before or after planting. Method described.

15. The method according to claim 12, wherein the mutant bacterium is introduced into seeds before or after planting. How to put it on.

16. The method of claim 12, wherein the bacteria are introduced as an aerosol or liquid. .

17. The bacteria have the ability to bind to lectins produced by legumes. 13. The method according to claim 12.

19. The gene was transformed into Rh1zobiuo+trifolium and Rhiz 19. The method according to claim 18, obtained from Obium leguminosarum.

20. A fixed nitrogen source is provided by the method according to any one of claims 12 to 19. The plants that were harvested.

21, mutation n1 for insertion by gene replacement into the recipe endobacteria fL gene, and as of August 30, 1991, the National Co11ection of Industrial and MarineB acteria Ltd,, ^berdeen, 5cotland, UK The yin61andii strain deposited with accession number 40438, or the same derived from its mutants or variants with the same growth and ammonia production functions. A gene characterized by

11.7.1N No. PCT/G89210036811.1A, l11. Pottery ρ Continuation of CT/G892100368 front page (51) Int, C1,6 identification symbol, internal office reference number C12R1:065) (72) Inventor Kennedy, Christina Kay United States, Alisina 8 1141 East Deer Canyon Road, Tuscon, 5718 I

Claims (22)

[Claims] 1. Nitrogenase activity is controlled by nifA or nifA-like genes Furthermore, nifA or nifA-like gene is nifL or nifL-like gene. A mutant nitrogen-fixing bacterium that is regulated only by functional nifA or is a mutation in the nifL or nif1-like gene rather than the nifA-like gene. Bacteria characterized by the degeneration of having 2. The bacterium according to claim 1, which belongs to the genus Azotobacter. 3. Claim 2, which is of Azotobacter vinelandii species. Bacteria described. 4. As of August 30, 1991, the National Collection on of Industrial and Marine Bacteria Ltd. , Aberdeen, Scotland, UK accession number 40 438, or its variant with the same ammonia overproduction function. 4. The bacterium according to claim 3, which is a natural mutant or variant. 5. A method for producing the mutant bacterium according to any one of claims 1 to 4, comprising: Nitrogenase activity is controlled by nifA or nifA-like genes. , furthermore, njfA or nifA-like genes are only nifL or nifL-like genes. isolating the nifL gene of the parent nitrogen-fixing bacterium, which is regulated by mutating the nifL gene; and mutating the nifA or nifA gene. While preserving the functionality of the similar gene, the nifL gene of the parent nitrogen-fixing bacterium was mutated as described above. and replacing with a different nifL or nifL-like gene. . 6. The nifL gene from one bacterium (recipient) is transferred from another bacterium (donor) 6. The method according to claim 5, wherein the mutant nifL gene is replaced with a mutant nifL gene. 7. The donor and recipient bacteria are of the same genus or family. The method described in claim 6. 8. 8. The method according to claim 7, wherein the donor and recipient bacteria are of the same species. Law. 9. 7. The donor and recipient bacteria are Azotobacter. The method described in. 10. The donor and recipient bacteria are Azotobacter vinyl 9. The method according to claim 8, wherein the andii. 11. The said donor and recipient bacteria are as of August 30, 1991 National Collection of Industrial a nd Marine Bacteria Ltd. , Aberdeen, S cotland, UK under accession number 40438, or the same strain. A claim that the invention is a mutant or variant thereof that has growth and ammonia production functions. 8. The method described in 8. 12. 5. A method of providing a fixed nitrogen source to plants, the method according to any one of claims 1 to 4. or according to any one of claims 5 to 11. A method consisting of introducing mutant nitrogen-fixing bacteria into nitrogen-fixing symbiosis with plant tissues. . 13. 13. The mutant bacterium according to claim 12, wherein the mutant bacterium is introduced into soil before or after planting. How to put it on. 14. 13. The mutant bacterium is introduced into the plant before or after planting. Method described. 15. 13. The mutant bacterium is introduced into seeds before or after planting. How to put it on. 16. 13. The method of claim 12, wherein the bacteria are introduced as an aerosol or a liquid. . 17. The bacteria have the ability to bind to lectins produced by legumes. 13. The method according to claim 12. 18. The gene that confers the ability to bind to lectins was found in Azotobacteria. 13. The method according to claim 12, wherein the method is introduced into A. vinelandii. 19. The gene was isolated from Rhizobium trifolium and Rhizobium trifolium. 19. The method according to claim 18, obtained from Biumleguminosarum. 20. A fixed nitrogen source is provided by the method according to any one of claims 12 to 19. The plants that were harvested. 21. mutation ni for insertion into the recipient bacterium by genetic replacement. fL gene. 22. As of August 30, 1991, the National Collect ion of Industrial and Marine Bacteria Ltd. , Aberdeen, Scotland, UK with accession number 404 A.38 deposited in vinelandii strain, or the same growth and characterized by being derived from a mutant or variant thereof that has a near-producing function The gene according to claim 21.