NZ241826A - Nitrogen fixing bacteria with mutation in nifl gene and its preparation and uses - Google Patents

Description

24 1 82 6 o Priority Datc-(s): CoiTipSeto Clace; C&M&kl, 0/f\ C'MV^ . flo hyp's ioo Public- 2 6 MAY 1993 ~ NEW ZEALAND PATENTS ACT, ] 953 No.: /"/We, ANIL KDMAR BALI, a citizen of India, of 1538 Trinagar, Nr. Canara Bank, Delhi 110035, India; GONZALO BLANCO, a citizen of Spain, of Bque, Anaso 2, 3C, Monte Quinto, Dos Hermanas, Seville, Spain; and CHRISTINA KAY KENNEDY, a US citizen, of 1141 East Deer Canyon Road, Tucson, Arizona 85718, United States of America hereby declare the invention for which X / we pray that a patent may be granted to aw/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by page la) r> 24182 - ta- 134719 o ^ AMMONIA PRODUCTION Field of the invention This invention is in the field of ammonia production by 5 nitrogen fixing bacteria.

Description of the prior art Many microorganisms are able to assimilate the major bioelements (i.e. carbon, nitrogen, sulphur, hydrogen and oxygen) in an inorganic form. The ability to use N2 as a 10 nitrogen source is restricted to prokaryotes, and 1s relatively rare even among this group. The enzyme system responsible for N£ fixation is called nitrogenase. Biosynthesis of the components of the nitrogenase system is determined by 15 to 20 different nvf genes.

Free-living nitrogen fixing bacteria fix an amount of atmospheric nitrogen sufficient for their own needs. Evidence for this is that significant amounts of ammonia are rarely found in the culture medium (in laboratory cultures) or environment (in the soil) of nitrogen fixing organisms. Regulatory 20 mechanisms controlling nrf gene transcription and/or nitrogenase w activity ensure that cellular energy is not wasted by the fixation of more nitrogen than is necessary to meet the demands of bacterial cell growth and viability. In particular, excess environmental ammonia or oxygen prevents expression of nif genes 25 in free living diazotrophs (nitrogen fixing bacteria). Ammonia makes nitrogenase unnecessary and oxygen inactivates the enzyme. Since these bacteria produce ammonia for assimilation in the form of ammonium cations (NH4+), the term "ammonium" is used hereinafter for brevity.

Attempts to induce ammonium excretion have up to now centered on physiological suppression or genetic manipulation of the enzymes Involved in ammonium assimilation. Treatment of cyanobacteria with L-meth1on1ne-DL-sulfoxim1ne (MSX), an inhibitor of glutamine synthetase (GS), resulted 1n the 35 excretion of 0.3 to 7 mM NH4+ into the growth medium, (Musgrove o V*jejr o ;- 2 - ;£i al-, 1982, Biotech. Letters, 4, 647-652, Newton et al-, 1985, Biochim, Biophys. Acta, 1&2, 44-50 and Ramos £± al., 1984, Appl. Environ. Microbiol., 4£, 114-118). Mutants of Anabaena resistant to MSX or the NH4+ analogue ethylene diamine excreted 5 up to 1.6 mM NH4+, (Polukina ai al., 1982, Microbiology, 51, 90-95, Spiller si. al-, 1986, J. Bacterid., 165, 412-419 and Thomas ft al., 1990 Appl. Environ Microbiol., 5£, 3499-3504). Among eubacteria, ammonium excretion was reported to occur in Gin" or Gin" asm-1 mutants of Klebsiella pneumoniae. (Anderson 10 ai al-, 1977, J.Gen. Microbiol., 102, 107-122 and Shanmugam £ial-, 1975, Proc. Natl. Acad. Sci. USA. 72, 136-139), in mutants of Rhodobacter capsulatus and Azospirillum brasilense altered in the production of GS, (Wall £l al-, 1979, 0. Bacteriol., 122, 1459-1463), and in methylamine resistant 15 mutants of Azotobacter vinelandii. (Gordon a± al., 1983, Can. J. Microbiol., 2f), 973-978). Although significant ammonium excretion has been observed, these chemically treated or mutated organisms require supplementation of large amounts of the amino acid glutamine for growth. ;20 The regulatory pathways and mechanisms Involved 1n the repression of njf genes by ammonium have been extensively characterized in the free living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii. Of central importance 1n both organisms (and other gram negative nitrogen fixing 25 bacteria) is that a positively- acting regulatory protein NIFA is required to activate transcription of the other nif genes which are necessary for nitrogenase structure and activity. In K. pneumoniae, ammonium represses nitrogenase synthesis by preventing either the activity or synthesis of NIFA, which 30 occurs by two separate mechanisms: The nifA gene is adjacent to and downstream of nifL (thus forming the nifLA operon) and these two genes are co-expressed. The NIFL protein binds to and inactivates NIFA 1f ammonium 1s present even at relatively low levels (> approx. 5pM). At higher levels of ammonium (> approx. 35 200nm), expression of the nifLA operon does not occur and so the ;n n ;© ;24 1 8 ;- 3 - ;NIFA protein is not synthesized. Transcription of nif LA requires phosphorylated NTRC protein but this protein is dephosphorylated and hence inactive in cells grown with ammonium. Thus in K. pneumoniae, repression of nitrogenase 5 synthesis by ammonium occurs at two levels, inactivation of NIFA by NIFL and prevention of nifA expression by dephosphorylated NTRC. ;Nitrogen fixation in A. vinelandii is determined by the presence of three biochemically and genetically distinct 10 nitrogenase enzymes, each of which is synthesized under different conditions of metal supply. The molybdenum nitrogenase which is similar to the enzyme purified from a number of other nitrogen fixing organisms, requires 15-20 gene products for its structure and activity. In A. vinelandi i as in 15 K. pneumoniae expression of the nif genes requires active NIFA product. Unlike in K. pneumoniae however, NTRC is not required for nitrogen fixation which implies that NTRC is not necessary for nifA expression. ;DNA sequencing of the nifA region of A. vinelandii revealed 20 a gene whose translation product was similar to NIFA of K. pneumoniae. The DNA sequence of 300bp upstream of nifA revealed a partial open reading frame (ORF) which predicted a protein with homology to the C-terminal regions of the nifL and ntrB gene products of K. pneumoniae. (Bennett £l al-, 1986, Mol. 25 Microbiol., 2, 315-321). ;It would be desirable to be able to induce ammonium production in these nitrogen fixing bacteria for a variety of purposes. Attempts to de-regulate nitrogenase synthesis in Klebsiella pneumoniae by mutating either the nifL or ntrC genes 30 were unsuccessful. The resulting nifL mutant was only weakly able to escape ammonium repression, and the ntrC mutant was nifA-, which means that nitrogenase synthesis does not occur and the organism is unable to fix nitrogen at all. ;The problem is that attempts to produce ammonia from 35 nitrogen fixing bacteria by de-regulation of nitrogenase ;o ;24 18 ;- 4 - ;production have been unsuccessful and although attempts to produce ammonia by genetically manipulating ammonium assimilation enzymes have been successful, the organism itself is unable to grow normally. ;5 Summary of the Invention ;It has surprisingly been found that in organisms wherein nitrogenase synthesis is controlled by nifA or n1fA-1 ike genes which are in turn regulated only by nifL or nifL—1 ike genes, mutations in the nifL or nifL-1ike genes result in mutant 10 strains of bacteria which produce and excrete significant amounts of ammonium. Surprisingly, these mutants grow as well as the parental strains. ;Description of the preferred embodiments ;This invention is applicable to any species of nitrogen 15 fixing bacteria which contain nifL or nifL-1ike genes as part of their nitrogenase enzyme system, said nifL or nifL-1ike genes solely controlling the transcription of nifA or nifA-like genes which in turn regulate transcription of other nif genes necessary for nitrogenase structure and activity. For 20 convenience hereinafter, nifL or nifL-1ike genes will be referred to simply as nifL and likewise nifA or nifA-like genes will be referred to as nifA. ;According to a first aspect of the invention there is provided a mutant of a nitrogen fixing bacterium as described 25 above, characterised in that it contains a mutant nifL gene and a functional nifA gene. ;The preferred bacteria are those which normally have strong plant associations, either by being present in the rhizosphere or by being attached to plant roots or living inside plant roots 30 or stems (so called systemic endophytes), or all bacteria from the genus Azotobacter whether plant associated or not. Particularly preferred bacteria are from the genus Azotobacter. especially Azotobacter vinelandii. The most preferred strain 1s the subject of a patent deposit, deposited 1n accordance with 35 the provisions of the Budapest Treaty at the National Collection ;24 1 8 ;- 5 - ;of Industrial and Marine Bacteria Ltd., (NCIMB), 23 St. Machen Drive, Aberdeen, AB2 1RY, Scotland, United Kingdom, on 30 August 1991 and given the accession number 40438, or a mutant or variant thereof having the desired functions of growth and 5 production of ammonia. ;According to a second aspect of the invention, there is provided a process for the isolation of the nifL gene by, for example, standard heterologous hybridisation techniques, from the nitrogen fixing bacteria described hereinbefore, the 10 construction of the mutations in the nifL gene, and the reintroduction of nifL mutations into the chromosome of the bacteria to replace the wild-type nifL gene. It 1s necessary that mutations inactivate the function of the nifL gene product but do not interfere with expression of the downstream nifA gene 15 (if it is located downstream). Examples are presented which show that nifL mutants continue to produce nitrogenase and express nitrogenase genes (nifHDK) in the presence of high levels of ammonium in the medium. ;It will be apparent to those skilled in the art that 20 mutations may be carried out in a variety of ways. The mutations may be carried out by the addition of extra genetic material into the nifL gene (so called "insertion mutants") by combination of deletion and insertion, deletion of part of the nifL gene (so called "deletion mutants") or exchange of nucleic 25 acid sequences rendering the protein non-functional for those rendering the protein functional. Such exchanges may be as large as the nifL gene itself, or an exchange of a single base in the DNA coding for the gene (so called "point mutation") may be sufficient. Techniques for carrying out the mutations will 30 be apparant to those skilled in the art. ;In the preferred bacteria of the invention, the nifL and nifA genes are normally found in an operon, under the control of one promoter, with nifL transcription occurring prior to nifA transcription although in other organisms this may not be the 35 case. The functionality of the nifA gene must be preserved in ;24 1 8 ;o ;© ;- 6 - ;the mutant. That Is, the nifA gene must be retained as in the parent organism or, if altered, the alteration must be non-interfering. It is important that the mutation does not prevent nifA production, for example, by insertion of a point 5 mutation into nifL which introduces a termination signal or by introduction of a polar mutation. The mutation must therefore be one which does not interfere with mRNA production. The nifA gene has its own Shine-Dalgarno sequence and hence the mutation in nifL need not be in-frame with the reading frame of nifA. 10 since transcription of nifA will continue as normal, despite mutation of nifL. It is known that the nifLA operon promoter is such that nifA is produced at levels which the bacteria find physiologically tolerable. Over-production of nifA by, for example, mutations which result in the production of a strong 15 promoter, can be detrimental to the cell. Therefore mutations in the nifL gene must not result in replacement of the natural gene promoter with a stronger promoter. The promoter of the nif LA operon must not be stronger than the natural promoter found in the wild type bacterium and preferably is the natural 20 promoter. ;When carrying out the invention in the deposited strain of Azotobacter vinelandii it is therefore preferable, in order to achieve normal expression of nifA in the nifL mutant, to retain a 200 base pair region between the Balll and Smal sites upstream 25 of the nifL coding region. This region carries the promoter of the nifLA operon in the A. vinelandi 1 strain. ;According to a third aspect of the invention there is also provided a mutated nifL gene which may be inserted into wild type bacteria In order to render them nifL-. Preferably the 30 mutant gene is from Azotobacter and 1s Inserted into other Azotobacter species. More preferably, the gene 1s from the deposited strain of Azotobacter vinelandii. ;According to a fourth aspect of the invention there is provided a method of controlling the level of ammonium 35 production by bacteria described hereinbefore. It 1s known that ;24 1 82 ;- 7 - ;the increase in pH of the culture medium which occurs concomitantly with ammonium excretion, limits the amount of ammonium excreted because the high pH inactivates nitrogenase. Control of pH, either by increased buffer capacity of the medium or by removal of the ammonium as it is excreted, can be utilized to give higher levels of ammonium production, or lower levels as required, for example in the use of nifL mutants of A. vinelandii. or other nitrogen fixing organisms described hereinbefore to produce ammonium whilst cells are immobilized on calcium alginate particles or beads. ;The invention is particularly useful for those nitrogen fixing bacteria which are normally abundant in the rhizosphere or found associated with specific plants, either by being bound to glycoproteins such as lectin on plant roots or actually living inside plant roots and stems (systemic endophytes). ;According to a fifth aspect of the invention, there is provided a method of providing plants with a source of fixed nitrogen by introduction of mutant nitrogen fixing-bacteria as described hereinbefore into nitrogen-fixing association with plant tissue (including parts of plants, seed, whole plants etc.). Nitrogen-fixing association with plants can manifest itself in several ways. Once introduced into nitrogen-fixing association with plants the bacteria may be present in the rhizosphere, or be associated with plants as described above. The mutant nitrogen fixing bacteria may be introduced to the plant tissue in a variety of ways. They can be applied to the plants themselves before or after planting, the soil before or after planting has occurred or to seeds. Conveniently the bacteria are applied as an aerosol or in a liquid or solid form. ;The mutant bacteria may be of a totally different family, genus or species to the wild type bacteria normally found in nitrogen-fixing association with plants. ;The mutations 1n the nifL gene may be introduced by mutating the wild type bacterium itself by methods hereinbefore described or may be introduced by gene replacement. The bacteria may be ;mutants of the wild type bacteria which are normally found associated with plants. Gene replacement involves the replacement of a gene from one organism with, in this case, a mutant nifL gene from another organism. Preferably, the two organisms are of the same family or genus, more preferably of the same species or strain. ;The invention also provides a way of introducing a source of fixed nitrogen to those plants to which there may be no nitrogen fixing bacterium normally associated. Preferably the mutant bacteria providing the source of ammonium to these plants is Azotobacter vinelandii. which is able to bind to lectins present on plant roots. Such Azotobacter vinelandii capable of lectin binding is constructed according to the teaching described by Bishop e± al-. 1977 Science, ]j)£, 938-939. These papers describe the construction of A. vinelandii strains that bind to lectins produced by a species of leguminous plants, Trifolium repens. The genes important for providing the ability of lectin binding will be transferred from either Rhizobium trifolium or R. leauminosarum to A. vinelandii nifL mutant strains. The gene for lectin production from Pisum sativa will be transformed into plant cells of a variety of species as described in Diaz al-, Nature, 1989, 223., 579-581. The production of P. sativa lectin receptors in A. vinelandii will allow 1t to bind to roots or other tissues of plants carrying genes that determine lectin binding from Rhizobi um leauminosarum. The binding of A. vinelandii nifL mutants to plant roots or other tissues may allow the provision of fixed nitrogen from bacteria directly to plants. ;Description of the drawings ;Figure 1 shows a restriction map of the Azotobacter vinelandii nifLA operon, and construction of plasmids. ;Figure 2 shows the levels of ammonia production by mutant and wild type Azotobacter vinelandii. ;Embodiments of the invention will be described by way of Example only. ;24 1 8 ;o ;O ;- 9 - ;EXAMPLE 1 : Growth of A. vinelandii ;Strains of A. vinelandii were grown aerobically at 30°C in Burk's sucrose medium as described previously by Toukdarian £i Al., 1986, Embo J., 5, 399-407. Liquid 25ml cultures, contained 5 in 125ml flasks, were incubated on a rotary shaker (180rpm). Competence medium (CM) was Burk's sucrose medium prepared without the addition of Fe and Mo salts. LB medium was used for growing E. col 1. Antibiotics for selection of resistance genes on plasmids or in genomic transformants were added at 10 concentrations previously reported (Santero Al-, 1988, Mol. Microbiol., 2, 303-314). ;EXAMPLE 2 : Isolation of an A. vinelandii genomic fragment carrying the region upstream of nifA ;About 300bp of the 3'-end of a gene encoding a protein with 15 partial sequence homology to the C-termini of the nifL and ntrB gene products of K. pneumoniae had been cloned in the plasmid pDB150 and sequenced by Bennett £t a1-, 1988, Mol. Microbiol., 2, 315-321. In order to isolate and clone the entire upstream gene, plaques from a lambda library of A. vinelandi1 genomic DNA 20 were screened for hybridization to a DNA probe labelled with 32p_ddp. This probe was a 1.2Kb SalI-Kpnl fragment from pDB150 which contains the 3' —end of the nifL/ntrB-1 ike gene and the 5'-end of nifA (see Fig. 1) (Bennett £± Al-, 1988, Mol. Microbiol., 2, 315-321). An approximately 12.5Kb Es&RI fragment 25 was identified in the insert DNA prepared from the progeny of ^ one hybridizing plaque; it was subcloned into pBR325 to give pAB21. (This 12.5Kb fragment corresponded 1n size to a genomic fragment previously reported to hybridize to a nifA probe; a nifA::Tn5 mutant, MV3, contained a new nlfA-hybrldizing fragment 30 of about 19Kb in size (Santero £± Al-, 1988 Mol. Microbiol, 2, 303-814). A restriction map of the insert in pAB21 showed that one end of the 12.5KB EcoRI fragment had restriction sites corresponding to those reported by Bennett £± Al- (see above) for pDBl50. Part of this fragment and the nlfA/nlfB region, and 35 subclones derived for this work, are shown in Fig. 1. ;n ;- 10 - ;Methods for blotting and screening the lambda library and for cloning DNA fragments were standard procedures described in Sambrook ii., 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, and by instructions 5 provided by suppliers of restriction enzymes. ;EXAMPLE 3 : Transformation of A. vinelandii ^ Competent cells were prepared by a simplification of a method described previously (Page fit al-, 1978, Can. J. Microbiol., 24 1590-1594): instead of growing cells in liquid 10 competence medium (CM) prior to transformation, cells were resuspended directly from the second round of growth on CM agar into 1ml of liquid CM + 16mM MgS04 to a density of about 108 cells ml-1. Approximately 50^1 of resuspended cells were spotted onto a CM agar plate and 0.1-l>ig of plasmid DNA was 15 mixed with the cells. After incubation at 30° for two days, approximately 5 x 10^ cells were transferred to selective medium containing appropriate antibiotics. Cells not mixed with DNA were plated as controls. Transformants arose at 104 - 10^ (jig DNA)"1. ;^ 20 EXAMPLE 4 : Isolation of the KIXX Cassette ;^ The region upstream of nifA in A. vinelandii was identified and cloned as described in Example 1. The KIXX cassette, containing the KMr gene (aph) from Tn5, was isolated as a 1.5Kb Smal fragment from the plasmid pUC4-KIXX (Pharmacia Ltd., UK). _ 25 This fragment was ligated with pAB26, which had been digested w with Sail and Smal: the Sail overhang was blunt-ended using ;Klenow polymerase and deoxynucleotides (F1g. 1). The resulting plasmlds pAB29 and pAB30, with the KIXX cassette inserted in opposite orientations, were transformed as described 1n Example 30 4 Into two strains of A. vinelandii. wild type UW136 and the n1fH-lacZ fusion strain MV101. Kmr transformants were screened for resistance to carbenicillin (Cbr); CBS derivatives were assumed to have arisen from a double crossover recombination event in which the wild-type n1fL/ntrB-11ke gene was replaced by 35 the KlXX-containing DNA. Kmr CBS transformants of UW136 were ;o ;4 1 82 ;- 11 - ;obtained with pAB29 but not with pAB30. The pAB29-der1ved strain, MV376, was Nif+. Southern blots of DNA from MV376, MV378, and MV380, digested with Sail, were hybridized to a 32P-1abelied probe from the mutated region (the 0.3Kb Sall-SphI 5 fragment from pAB31; see Fig. 1). Southern hybridization experiments showed that in all three mutants, the 3.5Kb wild-type Sail fragment was absent and replaced by a 6.2Kb hybridizing fragment. ;Although the KIXX insertion in both orientations in the nifL 10 gene of A. vinelandii resulted in a Nif+ phenotype, insertions of Tn5 or the fi (transcription terminator) cassette in A. vinelandii nifL resulted in a Nif" phenotype. Therefore, the nifLA genes are probably cotranscribed in A. vinelandii as in K. pneumoniae. Although the KIXX &£h cartridge inserted upstream 15 to oppose transcription can inhibit expression of downstream genes in A. vinelandii the nifA gene in the nifL (KIXX) mutants MV376 and MV378 is obviously expressed. This could arise from unexpected promoter activity in the cassette or from promoter-like sequences generated by the KIXX insertion In the 20 nifL region. ;O Insertion of KIXX in the orientation where expression of A£h was in the same direction as nifA resulted in somewhat higher constitutive levels of |3-galactos1dase (MV380) than when inserted in the opposite direction (MV378). However, it. was not 25 possible to construct a Nif+ nifL2-KIXX derivative in the wild-type equivalent to MV380, which, as 1t carries a n1fH-lacZ fusion, is Nif". Therefore excessive expression of nifA driven by the very strong a£h promoter is probably lethal In a N1f+ A. vinelandii background, possibly due to diversion of too much ATP 30 towards nitrogenase synthesis and activity or to NH4+ toxicity. EXAMPLE 5 : Enzvme assays ;Nitrogenase and (5-galactosidase activities were measured as described previously. (Sambrook si al., 1989, "Molecular Cloning". Cold Spring Harbor Laboratory Press, Cold Spring 35 Harbor, and Walmsley ei al-, 1991, Appl. Environ. Microbiol., ;ft ;24 1 82 ;- ;12 - ;o ;5Z, ;522-524). ;The results of these assays are shown in Table 1 below:- ;TABLE 1 . ;5 ;Nitrogenase and (5-galactosidase activities in wild-tvpe and nifL mutants of A. vinelandii o ;10 ;Strain Genotvpe ;UW136 wild-type MV376 nifLI:KIXX ;di +nh4+ ;48a 0 48 46 ;15 ;MV101 nifLl:lacZ ;15841b 432 ;20 ;MV378 nifHl:lacZ nifLl:KIXX ;MV380 nifHl:lacZ nifL2:KIXX ;11967 3772 15384 6474 ;25 ;-n ;N free medium. ;a ;Nitrogenase activity in ;UW136 ;and MV376: measured as o ;acetylene reduction (nmol ethylene produced min-' (mg ;protein)-1). ;30 ;b ;(3-galactosidase activity in Mi Her units (26). ;MV101, ;MV378, MV380: measured as v "s ;35 ;40 ;Each value is the mean from 2-3 independent determinations; variation was < 10% from the mean. ;The data in Table 1 show that MV376 was Nif+ and expressed wild-type acetylene reduction activity both in N-free medium and in medium with ammonium at 15mM, a concentration that repressed nitrogenase activity in the wild-type strain UW136. Similarly, both MV378 and MV380 expressed (3-galactosidase activity in the absence or presence of ammonium whereas activity in the nifL* strain MV101 was repressed by 15mM ammonium. 24 18 o r ) Other activities repressed by ammonium in A. vinelandii are those of nitrate reductase and nitrite reductase, (Luque si al-, 1987, Arch. Microbiol., 14£, 231-235), the expression of which requires both the ntrC and rpoN gene products, (Luque £± al., 5 Supra, Santero al., 1986, 0. Bacterid., 1M, 541-544 and Toukdarian §± al., 1986, EMBO 0., 5, 399-407). The KIXX insertion mutation had no effect on normal regulation of nitrate reductase; this activity was present in N03~-grown but not NH4+(+ N03~)-grown cultures of both wild-type UW136 and mutant 10 MV376 (data not shown).

These results demonstrate that the gene upstream of nifA in A. vi nelandi i mediates ammonium repression of nif gene expression but not of at least one other gene under ammonium control. This phenotype is somewhat similar to that of nifL 15 mutants of K. pneumoniae where nifL is located immediately upstream of nifA. although the escape of nitrogenase synthesis from ammonium repression is far greater in the A. vinelandii nifL mutants than in the K. pneumoniae nifL mutants. Translation of the entire A. vinelandii upstream gene sequence ^ 20 (approximately 1.4kb) shows a protein with greater homology to K. pneumoniae nifL, than to NTRB (31% vs. 24% identity).

EXAMPLE 6 : Ammonium estimation Culture supernatants of A. vi nelandi i wild-type strain UW136 and mutant strain MV376 grown on nitrogen were tested for the 25 present of ammonium.

Samples of cultures were taken at different times and centrifuged. 0.5ml of supernatant or filtrate was tested for the presence of ammonium by the indophenol method, (Bergersen, 1980, "Methods for evaluating biological nitrogen fixation", 30 John Wiley & Sons Ltd., London). This consisted of the addition, in order, of a) 0.5ml phenol-sodiurn nitroprusside solution (phenol, 50g l"1 + sodium nitroprusside, 0.25g l-1); b) 0.5ml of sodium hypochlorite solution (0.1m); and c) 2ml of distilled water. The mixture was incubated for 30 min at room 35 temperature. Absorbance at 625nm was measured and ammonium 24 182 concentration estimated from a standard curve obtained with ammonium solutions at various concentrations assayed using the same reagent solution.

The results are drawn in Figure 2, wherein • denotes UW136 5 and □ denotes MV376.

In contrast to UW136, MV376 excreted ammonium rather suddenly towards the end of exponential growth. Final amounts measured in MV376 stationary phase cultures were 5-10 mM. Excretion was not limited by carbon source availability because 10 half of the supplied sucrose remained at its cessation.

EXAMPLE 7 : Construction of nifL deletion mutant of A. vi nelandi i The plasmid pAB27 carrying the nifL region was digested with Ball I and li gated to a 3.8 Kb BamHI fragment from pJOl 7 which 15 carries the gene cartridge, (Hynes fit il., Gene, 1989 7£, 111-120. The ii£ gene confers a sensitivity to sucrose when transferred to certain gram negative bacteria, i.e. organisms carrying the nc gene cannot grow on sucrose-containing media. The resulting plasmid, pLA5:Sac-Km, carries the cartridge 20 which replaces a 585 base pair Balll fragment, deleting the 5' -end of the gene and 20 base pairs upstream from the translation start codon. In order to transfer the nifL:sac mutation from pLA5:Sac-Km into the chromosome, the plasmid was transformed into A. vinelandii UW136 and kanamycin-resistant 25 colonies were selected. These were found to be unable to grow on sucrose-containing medium but could grow normally on glucose as carbon source. One colony was purified several times and had a stable sucrose-sensitive, kanamycin-resistant phenotype. This mutant was MV399. MV399 had a N1f minus phenotype because the 30 cartridge Insert prevented expression of the downstream nifA gene which is essential for expression of nif genes.

In order to replace the n1fL:sac mutation with a nifL deletion mutation uninterrupted by DNA, pAB27 was digested with BailI and ligated to itself. Plasmid pLA4 was Isolated and 35 found to contain the 585 base pair BcQ.II deletion within the nifL gene. MV399 was transformed with pLA4 and colonies growing on sucrose-containing ammonium-free medium were selected. These Nif plus colonies were also kanamycin-sensitive indicating that the sac-Km cartridge had been replaced. One such colony was purified and tested for ammonium excretion. This strain, MV440, was found to excrete up to lOmM ammonium into the growth medium and the pH of cultures increased to about 8.5 when no further ammonium was excreted. Thus a deletion of the 5'-end of the nifL gene, which does not affect expression of the downstream nifA gene, leads to a mutant strain of A. vinelandii which excretes large amounts of ammonium similar to the phenotype of the nifL:KIXX mutant MV376 described previously.

Claims (26)

/ *£-v-v • a 24 t 8 WHAT //WE CLAIM O o - 16 - efc&$wy

1. A mutant nitrogen fixing bacterium wherein nitrogenase activity is controlled by a nifA or nifA-like gene which is in turn regulated solely by a nifL or nifL-1ike gene characterised 5 by the modification that it has a mutation in the nifL or nifL-1ike gene, but a functional nifA or nifA-like gene.

2. A bacterium according to Claim 1 which is of the genus Azotobacter.

3. A bacterium according to Claim 2 which is of the species 10 Azotobacter vinelandii.

4. A bacterium according to Claim 3 which is the strain deposited at the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 30 August 1991 under the Accession number 40438 or a mutant or variant thereof having 15 the same function of overproduction of ammonia.

5. A process for the production of a mutant bacterium according to Claim 1, 2, 3 or 4 comprising the steps of isolating the nifL gene of a parent nitrogen-fixing bacterium wherein nitrogenase activity is controlled by a nifA or nifA-like gene which is in 20 turn regulated solely by a nifL or nifL-1ike gene, mutating the nifL gene and replacing the nifL gene of a parent nitrogen-fixing bacterium by the mutated nifL or nifL-1ike gene, while preserving the functionality of the nifA or nifA-like gene.

6. A process according to Claim 5 wherein a nifL gene from one 25 bacterium, (recipient) is replaced by a mutant nifL gene from another (donor) bacterium.

7. A process according to Claim 6 wherein the donor and recipient bacteria are of the same genus or family.

8. A process according to Claim 7 wherein the donor and 30 recipient bacteria are of the same species.

9. A process according to Claim 7 wherein the donor and recipient bacteria are Azotobacter.

10. A process according to Claim 8 wherein the donor and recipient bacteria are Azotobacter vinelandii. 35

11. A process according to Claim 8 wherein the donor and a 24 1 8 o - 17 - recipient bacteria are the Azotobacter vinelandii strain deposited at National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 30th August 1991 under the Accession number 40438 or a mutant or variant thereof having 5 the same functions of growth and production of ammonia.

12. A method of providing plants with a source of fixed nitrogen '*■***} which comprises introducing a mutant nitrogen fixing bacterium according to Claim 1, 2, 3 or 4 or produced by a method claimed in any one of Claims 5 to 11 into nitrogen fixing association 10 with plant tissue.

13. A method according to Claim 12 wherein the mutant bacteria are introduced to the soil before or after planting.

14. A method according to Claim 12 wherein the mutant bacteria are introduced to the plant before or after planting.

15 15. A method according to Claim 12 wherein the mutant bacteria are introduced to the seed before or after planting.

16. A method according to Claim 12 wherein the bacteria are introduced as an aerosol or liquid.

17. A method according to Claim 12 wherein the bacteria are 20 capable of binding to lectins produced by leguminous plants.

O 18. A method according to Claim 12 wherein the genes providing ability to bind to lectin are introduced Into Azotobacterla vi nelandi i.

19. A method according to Claim 18 wherein the genes are 25 obtained from Rhizobium trifolium and Rhizobium leauminosarum. 3

20. Plants wherein the source of fixed nitrogen 1s supplied by a method according to any of Claims 12-19.

21. A mutant nifL gene for insertion into a recipient bacterium by gene replacement. 30

22. A gene according to Claim 21 characterised in that it originates from the strain of A vinelandi i deposited at the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 30th August 1991 under the accession number 40438 or a mutant or variant thereof having the same 35 functions of growth and production of ammonia. - 18 -

23. A mutant nitrogen fixing bacterium as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

24. A process as defined in claim 5 for the production of a mutant bacterium substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

25. A method as defined in claim 12 of providing plants with a source of fixed nitrogen substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

26. A mutant nifL gene as claimed in claim 21 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. A* )U 1 ' By pte?Their authorised Agftffi A.J. PARK & SON / *' •-> : It 1 w