Microorganisms and their use in agriculture

Abstract

A strain of Gluconacetobacter diazotrophicus (Gd) characterised by the presence of at least one nucleic acid sequence selected from SEQ ID NOS 1-10 or variants or paralogues thereof and/or the presence of a single plasmid of about 17566 bp in size. Such strains, exemplified by IMI504853, are useful in agriculture, in particular as they are able to colonise plants intracellularly.

Claims

1. A strain of Gluconacetobacter diazotrophicus (Gd) comprising nucleic acid encoding one or more expression products encoded by SEQ ID NOS 1 to 10 and a plasmid that is less than 27455 bp in size.

2. The strain of Gd according to claim 1, wherein the nucleic acid encodes at least three of said expression products.

3. The strain of Gd according to claim 1, wherein the nucleic acid encodes at least five of said expression products.

4. The strain of Gd according to claim 1, wherein the nucleic acid encodes all of said expression products.

5. The strain of Gd according to claim 1, which comprises one or more sequences with at least 90% sequence identity to any of SEQ ID NOS 1 to 10.

6. The strain of Gd according to claim 1, which comprises at least one of SEQ ID NOS 1 to 10.

7. The strain of Gd according to claim 1, which comprises at least three of SEQ ID NOS 1 to 10.

8. The strain of Gd according to claim 1, which comprises at east five of SEQ ID NOS 1 to 10.

9. The strain of Gd according to claim 1, which comprises all of SEQ ID NOS 1 to 10.

10. The strain of Gd according to claim 1, wherein the plasmid is in the range of 17.5 to 17.6 kbp in size.

11. The strain of Gd according to claim 1, wherein the plasmid is about 17566 bp in size.

12. A composition comprising the strain of Gd according to claim 1 and at least one agriculturally acceptable carrier.

13. The composition according to claim 12, which is suitable for coating a seed.

14. A seed having a coating thereon, wherein the coating comprises the strain of Gd according to claim 1.

15. A plant or seed comprising the strain of Gd according to claim 1.

16. The plant or seed according to claim 15, wherein the strain of Gd is located intracellularly within.

17. A method for producing a plant product, the method comprising growing the plant or seed according to claim 15 and obtaining the plant product therefrom.

18. A method for enhancing the nitrogen-fixing ability of a plant, said method comprising applying the strain of Gd according to claim 1 to the plant, an environment around the plant, or a seed that gives rise to the plant, such that the strain of Gd colonizes the plant.

19. The method of claim 18, wherein the strain is applied to a wound of the plant, a growth medium of the plant, or the surface of the seed.

20. A strain of Gluconacetobacter diazotrophicus (Gd) deposited with CABI in the United Kingdom under the Budapest Treaty with deposit accession number IMI 504958.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention will now be particularly described by way of example with reference to the accompanying figures which are described as follows:

(2) FIG. 1A: is a gel showing PCR products from a range of designed to be strain or species specific, including primer sets A-H in Table 3 (shown in lanes 4, 5, 6, 7, 8, 9, 12 and 13 respectively).

(3) FIG. 1B is a gel showing PCR products from a range of designed to be strain or species specific, including primer sets I-M in Table 3 (shown in lanes 4, 5, 9, 12 and 13 respectively).

(4) FIG. 2: is a gel illustrating PCR products using Primer set E following inoculation of reactions with 100 ng of (1) OSR var. Ability, (2) OSR var. Extrovert, (3) rice var. Valencia, (4) wheat var. Willow, (5) grass var. Aberglyn, (6) grass var. Dickens, (7) maize, (8) quinoa, (9) Arabidopsis var. Columbia, (10) barley var. Chapeaux, (11) grass var. Twystar, (12) grass var. J Premier Wicket, (13) potato, and (14) tomato. Lane (15) contains amplicon produced from 10 ng genomic DNA from Gd, (16) contains the no template PCR control, and the molecular weight marker at each end of the gel is Hyperladder 1 kb plus (Bioline).

(5) FIG. 3: is a gel illustrating the sensitivity PCR using Primer set B in reactions containing 100 ng DNA from OSR var. Ability, co-inoculated with (1) 1 ng, (2) 100 picogram, (3) 10 picogram, (4) 1 picogram, (5) 100 femtogram, (6) 10 femtogram, and (7) no added genomic DNA from Gd. Lane (8) is the no template control sample and molecular weight marker at each end of the gel is Hyperladder 1 kb plus (Bioline).

(6) FIG. 4A is a graph showing positive amplification of Gluconacetobacter diazotrophicus by fluorescent LAMP using the Genie II real-time machine and a primer set embodying the invention. Positive DNA amplification is detected by a fluorescence signal. FIG. 4B is an anneal curve for the Gluconacetobacter diazotrophicus samples, following amplification by LAMP; the reaction was put through an anneal analysis and the temperature at which the dsDNA reanneals is detected as a burst of fluorescence.

(7) FIGS. 5A-C are graphs showing representative results of QPCR experiments carried out using primers designed to amplify sequences according to the method of the invention, when carried out using serial dilutions of samples containing GD DNA. FIG. 5A shows the results for primer set designated P5 for SEQ ID NOs 58 and 59. FIG. 5B shows results for a primer set designated P8 for SEQ ID NOs 60 and 61. FIG. 5C shows results for a primer set designated P17 for SEQ ID NO 62 and 63 as defined hereinafter.

(8) FIGS. 6A-F are graphs showing representative results of QPCR experiments carried out using primers designed to amplify sequences that may be detected using a kit of the invention, when carried out using serial dilutions of samples containing GD DNA and plant genomic DNA. FIG. 6A shows the results for primer set designated P5 in the presence of wheat DNA. FIG. 6B shows melt peak graphs of the products of FIG. 6A for all the samples (i.e. dilutions of Gd in presence of wheat genome and relevant controls). FIG. 6C shows melt peak graph of the controls from FIG. 6A where a positive control comprising Gd DNA only resulted in giving signal, and negative controls comprising plant DNA only and QPCR negative samples only (NTCno transcript control), both did not resulted in giving signal. FIG. 6D shows results for a primer set designated P17 as defined hereinafter in the presence of maize DNA. FIG. 6E shows the melt peak graph of the products of FIG. 6D for all the samples tested (i.e. dilutions of Gd in presence of Maize genome and relevant controls). FIG. 6F shows the melt peak graphs of the controls from FIG. 6D where a positive control comprising Gd DNA only resulted in giving signal, and negative controls comprising plant DNA only and QPCR negative samples only (NTCno transcript control), both did not resulted in giving signal.

(9) FIG. 7 shows a resolved 1% agarose gel showing plasmid DNA extracted from a particular strain of GD (IMI504853).

(10) FIG. 8 shows resolved 1% agarose gel restriction digestion product of plasmid DNA with EcoRI from strain of Gd (IMI504853). The restricted fragments are mentioned as 1) 12 Kb and 2) 5.6 Kb when run alongside 1 kb ladder where the nearest fragment from the ladder is highlighted for the size comparison.

(11) FIG. 9 shows an agarose gel obtained from a PCR amplification to detect Gd from the seedlings of wheat obtained during a field trial.

(12) FIG. 10 is a graph showing the chlorophyll index of wheat treated with Gd in accordance with the invention compared to a control.

(13) However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The following descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. They are not intended to be exhaustive of or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

EXAMPLE 1

(14) Identification of Unique Sequences

(15) IMI504853, a Gd strain derived from passaging UAP5541, which was found to have particularly beneficial plant colonisation properties was isolated and the full genome sequenced. A comparison was made against the publically available genome of the type strain (PAL5; sequenced by JGI, USA [Genbank sequence accession CP001189]) using standard methods.

(16) Surprisingly, a large number of differences were noted in the genome, and in particular, a number of genes were identified which are present in the genome of IMI504853 but not PAL5.

(17) Many of these genes were annotated with an associated function. The unique genes with annotations were further checked for uniqueness across all the genomes sequenced to date using the NCBI's web-based BLAST tool.

(18) Analysis of the BLAST result narrowed the list to 20 unique genes not present in any genome. These unique genes appeared to be strain-specific for IMI504853.

(19) Also, five sets were found to be unique to the Gd species and will hereafter be referred to as species-specific (i.e. present in IMI 504853, Pa15 and other Gd strains but in no other species).

(20) Thus these sequence differences appear to characterise the strain and can be used to design a diagnostic kit for IMI504853 and similar strains.

EXAMPLE 2

(21) PCR Validation

(22) A set of 25 primer sets were designed based upon the sequences identified in the analysis of the genome. The specificity of these 25 primer sets (20 designed to be strain-specific and 5 designed to be species-specific) were first tested by carrying out a conventional PCR reaction using genomic DNA of IMI504853 and PAL5. The results with IMI504853 are illustrated in FIGS. 1A-B. Results showed that 16 strain-specific primer sets delineated IMI504853 from PAL5 as obtained from three different collections (ATCC49037, DSM5601 and LMG7603). However, 4 putative strain-specific primer sets cross-reacted with at least one PAL5 and hence were removed from strain-specific study.

(23) All 5 species-specific primers reacted as expected.

(24) Further, testing of strain- and species-specific primers was done against two other strains of Gd, one originally isolated in India (IMI 502398) and the other from Mauritius (IMI 502399), as well as a revived 2001 culture of UAP5541 strain (stored in glycerol at 80 C.), using the method described above. The data was in agreement with the 16 strain specific primers and 4 species specific primer sets as one of the species-specific primer sets produced a higher molecular weight band. This was a surprise result.

(25) The sensitivity of detection of all 25 primer sets (20 strain-specific and 5 species-specific) was checked using serial dilutions of bacterial broth cultures. It was found that 24 of these sets produced very high levels of detection (requiring 1-10 bacterial cells).

(26) Further, these 25 primers were then checked for cross-reactivity with several target plant species and varieties using DNA extracted in-house. The primers were tested in a PCR reaction using DNA extracted from plants of the following species: maize, wheat (var. Willow), quinoa, rice (var. Valencia), barley (var. Chapeaux), potato, Arabidopsis (var. Columbia), oilseed rape (vars. Ability and Extrovert) and a range of grasses (vars. Aberglyn, Dickens, J Premier Wicket, and Twystar). The method for isolating nucleic acids from plant tissues involved the mechanical maceration of leaf material followed by a modified CTAB extraction (Doyle and Doyle, 1987 Phytochem. Bull., 19: 11-15). Briefly, cellular membranes were disrupted using SDS and CTAB to release their contents, and cellular proteins were degraded or denatured using proteinase K and -mercaptoethanol. The extraction buffer also contained PVPP to remove plant polyphenols, EDTA to chelate metal ions, sodium chloride to solubilise nucleic acid structures, as well as TRIS HCl to stabilise the buffer pH. RNA molecules were degraded using RNase A treatment. Following the removal of insoluble cellular debris using chloroform:isoamyl mix (24:1), deoxynucleic acids were precipitated in ethanol using sodium acetate, washed using diluted ethanol, and resuspended in molecular grade water.

(27) Illustrative results are shown in FIG. 2.

(28) At the same time, the sensitivity of primers were tested by co-inoculating PCR reactions containing 100 ng of the above mentioned plant genomes with six-fold serially diluted genomic DNA from Gd, starting from 1 ng. It was found that the sensitivity of the PCR system was generally unaffected by the presence of plant genome and routine detection was established from a minimum of 1 picogram of Gd DNA.

(29) Illustrative results are shown in FIG. 3. Results suggested that 17 of the 20 strain-specific primer sets and three of the five species-specific sets either do not cross react with any plant genomes tested, or cross-react with a small number but produce a DNA product of a different size and distinguishable size.

(30) Of the strain-specific primer sets, only 10 produced results which were of (1) high specificity, (2) high sensitivity, and (3) produced no cross-reactions with plant DNA and these are represented in Table 3 above as primer sets A-J. Similarly only three of the selected species-specific primer sets were found to be specific and sensitive enough for use and these are shown as primer sets K-M in Table 3 respectively. In addition, the size of the products obtained using these primers is shown in Table 3 and illustrated in FIGS. 1A and 1B. Thus, methods and kits based upon these primers are particularly useful in identifying beneficial Gd in field situations.

EXAMPLE 3

(31) LAMP Assay

(32) A series of LAMP primers were designed to amplify regions of SEQ ID NOS 6, 7 and 9 and are shown in Table 4 below as follows:

(33) TABLE-US-00002 TABLE4 SEQ ID NO Sequence Type 40 CTCAGGAAGACCGAATTGATTA F3 41 GCGAAACGTCTGATTGAAC B3 42 CGGATAACCACTGGTGCTCCGACTCGCCTCACTCTACT FIP 43 TCCACGAATCTCACGAAGCACCCCGACCTTATCTCCCAT BIP 44 GCCAGGCGTGTACATATAACTA FL 45 CGGAATACCTAGTTGGAACACT BL 46 TCAAGATCGATGCACCTATTC F3 47 AACAGACAGTTCTGGTAGGA B3 48 CGCATCTCCAGATCGGCAGGTCGTCCAGTCGATCATG FIP 49 ACATCTGTCCACGGCATTGGTGGCTGGCTTATGAGTCT BIP 50 GAGAAGTCCTCTGCTTCGG FL 51 CGGCGGTTGAGAAGATGT BL 52 GGAAGACATCAACGAAGCA F3 53 TTGACAGTTGCATAGTCCG B3 54 ATACGGCTCGTCATGTCGCGGTGATGGATAATCTCAGCC FIP 55 CAGTGGCCGAACCTGGAAGCGCTGATATAAGCCTGAAGAT BIP 56 ATTGCACCGCGTTGATG FL 57 GCGTAACGGTCACAAGGA BL

(34) SEQ ID NOS 40-45 were designed to amplify SEQ ID NO 6 above, SEQ ID NOS 46-51 were designed to amplify SEQ ID NO 7 above, and SEQ ID NOS 52-57 were designed to amplify SEQ ID NO 9 above.

(35) These primers were obtained and tested in a LAMP assay on samples comprising pure Gd DNA that had been isolated using a modified CTAB methodology from bacteria grown in liquid culture.

(36) In addition, DNAs from a range of plant pathogenic bacteria and fungi was tested for amplification in LAMP by the primer sets. These included Bacillus subtilis, Lactobacillus, Fructobacillus, Pseudomonas spp., Agrobacterium spp., a range of phytoplasmas and various fungi including species from the Fusarium, Penicillium and Aspergillus genera.

(37) Real-time LAMP was carried out on a Genie II instrument (OptiGene), and 1 l of sample was added to a 24 l reaction mix containing 15 l Isothermal Master Mix ISO-001 (OptiGene), 200 nM of each external primer (F3 and B3), 2 M of each internal primer (FIP and BIP) and 1 M of loops primer (FLoop and BLoop). RT-LAMP reaction consisted of 30 minutes of isothermal amplification at 63 C. To evaluate the annealing temperature of the products, reactions were then subjected to a slow annealing step from 95 to 68 C. (0.05 C./s) with fluorescence monitoring.

(38) Negative reaction controls, consisting of water, were also used.

(39) Of the three sets of primers tested in LAMP, the third primer set specific for SEQ ID NO 9 gave amplification in nine and a half minutes with an anneal at 89.2 C. (see FIG. 4A). The primer set specific for SEQ ID NO 6 amplified the positive control at around 11 minutes with an anneal of approximately 88 C., and the primer set specific for SEQ ID NO 7 was the slowest, amplifying the positive control at around 23 minutes with an annealing temperature around 90 C.

(40) All sets of primers that gave the positive Gd amplification were specific for the bacterium and did not amplify from DNA of any of the other bacterial and fungal DNAs they were tested on. They are therefore all suitable as primer sets to be used for detection of the Gd bacterium.

EXAMPLE 4

(41) Detecting Gd on Plant Samples Using LAMP

(42) To validate the primers on rapidly extracted DNA from contaminated seed, a series of experiments were set up in which seed of two plant species, tomato and wheat, were spiked on the surface with Gd DNA. The samples were then put through the 2-minute DNA extraction technique in which the samples are placed in plastic tubes containing steel beads and TE buffer and shaken vigorously for 2 minutes. Two microliters of the solution was then placed in the LAMP reaction as described in Example 4 using the primer set comprising SEQ ID NOs 52 to 57 to test for amplification of the Gd DNA from these samples.

(43) The results showed that the Gd DNA is detectable when put through these assays, against a background of plant DNA.

(44) In order confirm that any samples that tested negative for Gd supported LAMP amplification (i.e. they do not contain inhibitors of LAMP reactions), the cytochrome-oxidase gene (COX) primers (Tomlinson et al., 2012 Journal of Virological Methods, 191: 148-154.), which amplify DNA from the host plant, were used as controls for false negatives on all samples.

EXAMPLE 5

(45) QPCR Determination

(46) A range of QPCR reactions were carried out on samples comprising known quantities of DNA from Gd (IMI504853) and also from a range of crop species including maize, barley and wheat genomic DNA.

(47) QPCR reaction mixtures were prepared to a volume of 20 L volume per reaction. In the case of Gd DNA alone, these consisted of 10 L iTaq Universal SYBR Green Supermix (2) (Bio-Rad), 1 L each of forward and reverse primers (final concentration of 10 mol), 7 L SDW (sterile distilled water) and 1 L DNA template at the required concentration.

(48) Primers used in this case were as set out in Table 5.

(49) TABLE-US-00003 TABLE5 SEQ ID NO Sequence Type 58 AGGAGGCTCTTTCTTTGGAAGC Forward 59 AAGTGCCCCTGTTATCGTACAC Reverse 60 TGGGTCATCGGTTCTGATTTCC Forward 61 TAGTTTGATGTCGGGTGCTGAG Reverse 62 GCGAATACCGGTCTTTTTACGC Forward 63 ATGCAAGCTCCGGATTGAGAG Reverse

(50) The primer set represented by SEQ ID NOs 58 and 59 (designated P5) was aimed at amplifying a 149 base pair region of SEQ ID NO 3, the primer set represented by SEQ ID NOS 60 and 61 (designated P8) was designed to amplify a 104 base pair region of SEQ ID NO 6 above, and the primer pair represented by SEQ ID NO 62 and 63 (designated P17) was designed to amplify a 130 base pair region of SEQ ID NO 10 above.

(51) Thermocycling was carried out using a CFX96 Touch Real-Time PCR Detection System from Bio-rad. Initial denaturation was performed at 95 C. for 3 minutes; amplification was performed using 40 cycles of denaturation at 95 C. for 5 seconds followed by 60 C. for 30 seconds (plate read post each amplification).

(52) All of the primer sets amplified Gd DNA with good efficiency as set out in Table 6, which shows the average Cq values of three replicates of the amplification, and quantitatively as illustrated by FIGS. 5A-C. The percentage efficiency was calculated using the formula % E=[10.sup.1/slope]1100.

(53) TABLE-US-00004 TABLE 6 P5 (SEQ P8 (SEQ P17 (SEQ Log ID NOs ID Nos ID NOs 62 + Dilutions 58 + 59) 60 + 61) 63) ng/l 1 19.23 19.68 19.96 12 2 22.75 22.55 23.39 1.2 3 26.19 26.10 26.78 0.12 4 30.23 29.97 30.75 0.012 5 33.64 32.85 33.96 0.0012 6 36.15 36.26 37.04 0.00012 7 NA NA NA Slope 3.4659 3.3613 3.4594 % Efficiency 94.32 98.38 94.57

(54) The quantitative amplification was carried out in the presence of genomic plant DNA in order to determine whether there was any cross reactivity. It was found that whilst there was cross reactivity with some plants species, the primer pairs P5 showed no cross-reactivity to wheat and barley genomes, P8 showed no cross-reactivity to wheat barley and maize and P17 showed no cross-reactivity to wheat and maize genomes making these potentially suitable primer sets for detecting Gd in crop species.

(55) To ensure that primer efficiency and robustness would be maintained in the presence of plant genomic DNA, the above QPCR examples above were repeated but in this case, the composition was varied in that 6 L SDW (sterile distilled water) was used together with 1 L relevant Gd dilution DNA template and 1 L plant genomic DNA template. For instance, Gd DNA (92 ng/l) was serially diluted from 10.sup.1 to 10.sup.7 with either wheat DNA (70.6 ng/l) or maize DNA (111 ng/l) and amplification reactions run as described above.

(56) Representative results are shown in FIG. 6A and FIG. 6C and in Table 7.

(57) TABLE-US-00005 TABLE 7 Gd + Plant DNA QPCR Std. curve Cq Value from QPCR run Log P5 (Seq ID P17 (Seq ID ng/l in dilutions 3)_Wheat 10)_Maize reaction 1 21.47 21.76 9.2 2 24.56 25.11 9.2 3 27.69 28.13 0.92 4 31.38 32.08 0.092 5 34.89 35.32 0.0092 6 37.55 37.80 0.00092 7 N/A N/A 0.000092 AzGd DNA 24.20 25.00 (0.01) Plant DNA N/A NA NTC NA NA Slope 3.2887 3.2794 % Efficiency 101.41 101.81

(58) It appears that the primers will maintain efficiency in the presence of plant genomes and thus may form the basis of a detection kit.

(59) Results were confirmed by carrying out melt analysis post amplification the denaturation curve (Melt curve) analysis was performed from 60 C. to 95 C. with 0.5 C. increment 5 seconds/step followed by plate read after each increment.

(60) Representative examples of the results are shown in FIGS. 6(B) and 6(E). Clear melt curves are visible for amplified Gd DNA, without plant genomic DNA.

EXAMPLE 6

(61) Plasmid Detection

(62) Plasmid DNA extraction from Gd (IMI504853) was performed using Qiagen mini prep kit (Cat. No. 69104). The low copy number plasmid extraction protocol was followed using 5 ml and 10 ml 48 hour bacterial culture. The extracted plasmid was run on 1% agarose gel flanked by a 1 kb ladder (FIG. 7) and imaged.

(63) Alongside the plasmid DNA, genomic DNA of Gd (IMI504853) was also included on lane-1. The results, shown in FIG. 7 indicate the presence of a single plasmid of about 17.5 Kb in size, which is smaller than that reported previously for plasmids found in PAL5.

(64) The plasmid DNA was sequenced and a primer was designed using Primer3 (Untergasser et al. (2012) Primer3new capabilities and interfaces. Nucleic Acids Research 40(15):e115; Koressaar and Remm (2007) Enhancements and modifications of primer design program Primer3 Bioinformatics 23(10):1289-91) to cover the start and end sites of linear sequence data (P_End_FwCCAAATCTCTGGAACGGGTA (SEQ ID NO 64). Sangar sequencing was performed using this primer (SEQ ID NO 64) and the sequenced data was aligned to confirm the plasmid sequence was complete.

(65) Since plasmid DNA in its natural form is circular and can form secondary and tertiary structures, this may impact on the accuracy of size measurements using agarose gels. To confirm the results and also validate the sequencing of the plasmid DNA, a restriction map of plasmid was studied using NEBcutter. The restriction digestion will linearize the plasmid providing only a single conformational structure. Also, the restriction enzyme selection is done after studying the sequence, thus allowing the plasmid sequence to be validated as well. In case of IMI504853 plasmid DNA, the NEB-cutter showed the restriction enzyme EcoRI to digest the plasmid DNA at 3864-9461 bp and 9462-3863 bp producing a DNA fragment of 5598 bp and 11968 bp. Both the size can be studied using a 1 Kb ladder available in the lab, removing the limitation of the reference ladder's maximum size detection as well.

(66) Therefore, restriction digestion was performed on IMI504853 plasmid using double cutter EcoRI (Fisher, cat no10819360) as per supplier's protocol. Post restriction digestion the products were run on 1% agarose gel until the bands were resolved and imaged. IMI504853 plasmid DNA when restricted with EcoRI produced two fragments (1. 12 Kb and 2. 5.6 Kb) of DNA of predicted size (FIG. 8).

(67) This validated the sequencing data in terms of both size and sequence. It may further provide an identification test or a confirmatory test in relation to a kit used in the identification of strains of the invention.

EXAMPLE 7

(68) Illustration of Activity of IMI504853

(69) A field trial was designed to test Gd (IMI504853) as a bio-fertilizer using wheat (cultivar Mulika). Two plots of Gd treated and control (untreated) respectively where planted. Post germination the young wheat seedlings at 10-12 day of growth were sampled and tested for Gd presence using the primer G (seq ID 26 & 27) representing the DNA seq ID 7. The Gd presence was detected when PCR was resolved on a 1% agarose gel with respective negative and positive controls (FIG. 9) confirming that in real world condition the designed kit works well.

(70) The measurement of chlorophyll content i.e. greenness using a SPAD meter has been shown to correlate with over all plant health and crop yield. The crop at growth stage 35 and 61 were checked for its chlorophyll content using SPAD was found to be statistically significantly (P=0.001) in Gd treated plots when compared to control plots (FIG. 10). Interestingly, the SPAD showed a significant increase in the chlorophyll content from the wheat obtained from plots treated with Gd of the invention compared to untreated controls (FIG. 10). This indicates that Gd treated plots which have been confirmed to have the bacterium present using the diagnostics kit results in much healthier plants and potentially higher yield. The data from wheat field trial indicates the efficacy of Gd as a bio-fertiliser.

EXAMPLE 8

(71) Yield Benefits of use of IMI504853

(72) Forage maize seed untreated and treated with a formulation comprising IMI504853 at a concentration of about 10.sup.5-10.sup.7 cfu/ml was sown at two different locations, and fertilised at different proportions of the recommended rate of nitrogen fertiliser, ranging from 0 to 175 Kg ha.sup.1 for each particular variety, region and availability of soil nitrogen. Yield results indicated an overall potential reduction in nitrogen fertiliser of between 40-95% without suffering any yield penalty. Average yield benefits across N levels led to an increase in yield of between 7% and 21%. Thus at full recommended N fertiliser rates IMI504853 treated crops could provide a yield benefit up to 1 t/ha in maize.

(73) In another trial, spring wheat seed, either untreated or treated with a formulation comprising IMI504853 at a concentration of about 10.sup.5-10.sup.7 cfu/ml was sown in Spring. The crop was fertilised at different proportions of the recommended rate of nitrogen fertilizer ranging from 0 to 125 Kg ha.sup.1 based on variety, region and availability of soil nitrogen. Across all fertilizer levels, IMI504853 treated crops showed an average yield increase of 15%, but at zero nitrogen fertilizer and full nitrogen fertilizer this increase was 20% and 10% respectively. The results indicate that for the IMI504853 treated crop, it is possible to reduce N fertiliser application by up to 85% and still achieve the same yield as a fully fertilised crop.

(74) TABLE-US-00006 TABLE1 SEQID1 GlutathioneS- ATGACAAAATTATACTATTCTCCCGGCGCTTGCTCTTTGGCAGGGCATATTTTGCTCGAAGAGTTGGGAAGACC transferase(EC ATATGAGTTGAAATTGACGCCCGTTGGAGACGAAGGCACGGGAAGTGAAGAGTTTCTAAAAATAAACCCGCGAG 2.5.1.18) GAAGGGTGCCTGTTTTAATTGATGGTGCGGAAATAATTACCGAAAGCCCCGCAATCTTATTTTATTTATCGAGT TCATTTTCAGACGGAAATTTCTGGCCAAAATCAGTTTTGGAGCAAGCCCGCTGCTGGGAATGGTTTAACTGGTT ATCGAGCAATGTACACTCGGTTGCCTATGGGCAGGTGTGGCGACCAGGACGGTTCATTGATGATGAGCGTCAGT GGAATAATGTTATTTCAAAAGGGAAAAATAACCTTCATGAATTTAGTGATGTAATAGAAAATAATATCTCCGGG AAAACGTGGTGTGTGGGTGAATCGTATTCATGCGTTGATCCGTATTTGTTTGTTTTTTATTCTTGGGGGAAAGC CATCGGATTGGATATGGAATCCTCTTTCCCGGCGTGGTCGCGTCATGCAGCGCGGATGCTGGAGCGGTTGGCCG TTCAAAACGCTTTACGGCAAGAAGGTTTGATCTCGTAA SEQID2 O- TTGGATGCCTCTCGTTTTCCTTGCGGAGTCATCATGACCATTCCTCTCTTTCGTCCGCAATTCACACCGCAGAT methyltransferase TCAACGTGCGCTTGATCGCCTTTATTCCGAGACACTCTCGCAAGATCCAGCGATACGCCAATTGGCGCAAGCCA (EC2.1.1.-) AAGGACTGACACATGACGGGCAACGCGGTTTCTACGAAGCCATGAAAGATGCCAGACTACCCGTTACGCCAGAG TTCGGCGCCCTGCTCTATATTCTGGCACGCAGCACCAGAGCCCAACATATCATCGAATTCGGCACGTCCTTCGG TGTTTCAACATTATTTCTCGCAGCGGCTTTACGCGACAATGGCGGGGGCCGACTGGTGACCTCGGAACTTATCT CAGACAAAGCAGAAAGGGCTTCCGCCAATCTGCGGGAGGCAGGACTGGCAGACCTCGTAGACATTCGCATCGGA GATGCCCGCGCCACGTTATCGCGTGATCTTCCTGAGTCGATCGATCTGATCCTGCTGGATGCGACTAAAGGACT CTACCTCGATCTCTTACTCCTGCTGGAACCTGCATTACGAAAAGGTGGCCTGGTGATCAGCGATCGCGCCGATC TCGATGGTGACGACGGCGGTCGCGCAGCAGCCTACCTTACCTATCTGACAACCCCGGCTAACGGATATCGCATC GCCGGCATCACTACACAGGCGTTGGGACAAACCTTCGCTCACGATGTGGCGGTGCGCACCTGA SEQID3 Transcriptional GTGGGAATAGCCACGCTCTACCAATACTTTGAGAACAAGGAGAGCGTTGTCGCGGCACTTAGTCGTCGGGTACG regulator,TetR GGAAACACTGCTCCATGATGTTGCGTCATTACTCGAAACCGCTTGTTCGTTGCCACTTTCCGAGGGTGTGCGCT family GTCTGGTCGTCGCTGCCGTGAAGGCGGACAAGAGCCGTCCATCGCTTACGGTCCGGCTTGATCGGTTGGAGGAG GCTCTTTCTTTGGAAGCGGATCATCTGCTGGTAGCGGCTGAGCTTTGCACGGTTGTTGCGTCGTTCCTCAAGTG CCAGGGAATTATTCAGGAGAACACTGCAAAAATTCTGGCAGATGATCTGTGTACGATAACAGGGGCACTTATTG ACGCTGCACACAATCGACAGATACCTATCGATGACTTGCTAATTGACCGTATTACGCGAAGGCTGGTCGCGATT ATTCAGAGCGCGCTTTAA SEQID4 RNApolymerase GTGGGACAGCCGGACAAATATTTCGAGCTTTTCGCGATACATCGCACCGATCTTGTGCGTTTCGCCAGAGGTAT ECF-typesigma CATGAGAGATGATAGTCTGGCGGAAGATGTTGTACAGGATGCTTTTCTGCGGCTGACTACTGTAACAGTGGCAC factor::RNA AGGACCGCGTTCTTTCGGATCCTCTGAATTACGTTTACCGCATTATTCGGAATCTGGCCTTTGACCGTTATCGA polymeraseECF- CGACGGCAATTCGAGGCCGGATTGTTTGACCATGGGGTAGATAGTTCTTCCGAAACAATCGAAGCGGATGCCCC typesigma TACACCGGAAGGTGAGGCTTCAGGGAAATCCGACATGCGGGCAATGCGCGCCGCTATGGCGGAACTGCCAGAAC factor GGACGTGCGTCGCATTGGAAATGCATCTGTTCGACGGACGAAAGCTACGGGAAATAGCGGCTCATTTAGGTGTT TCTATTGGGATGGCCCACTCCCTTGTCGCAGTGGGTATGGAGCATTGCCGCAAACGTCTTTCCACACCTGAAAC CTGA SEQID5 FecRfamily GTGAGCGAAGACTTCAATCCAACAACGGCGGTTGAGTGGAAAATAGCCCTTTCAGAAGAGCCTGACGATTCTGT protein, GCTCAGAGAACGCTTTGAAGCATGGCTTGCTGCCGCAGAGGATCACCGGAAAGAGTGGCAGGAACTTACGCAGG COG:Fe2+- GACTTGAGAATTTCCGCCAGATTGGCCCGCTTTATCGTGAGAAATGGGTGCCTTCATCAAGTGGGGCACAAAAT dicitrate ACTGCGTCAAAACAAGGTAGGCTCAAAGGAAGACCTGCTAATTTTGTTAGGTTTTCAGTTGCTGCATTTGCGGC sensor,membrane TGCTGCTGCCGTTACATTGGTATGGTCCTCTGACCTTCTGCTTCGATTACAGGCCGATTACGCCACAGGCTCGG component; CAGAAACGCGAACAGTCAGTCTCCCCGATGGTAGTGAATTGACCCTCGCGCCGCGAAGTGCGGTAAAAATGTCT TACTCTGTAGAGAAACGGGATATTCGTCTTTTAAAGGGAGAGGCGCTCTTCACGGTTCGACATGATATGGCGCG ACCTTTTGAGGTCCACACAGACAAATTCACCGTAACGGACATCGGAACTATTTTTGACGTCAGAATGTCTCAGG GCGAGGAAGAAGTCTCTGTCCGGGAAGGAGAGGTCCGGGTGCAGGATGTTTCCGGTGGATTTCATAATCTCGAT GCCGGAACGTGGGAGCGGATTAGAACTGTAGGCAATGGAGTGAGCGTCACTCATGGGAGCGGCTCTCCGGAAGA TGTGGGCGCATGGTCAGCGGGGCAAATTATTGCCAAGGAAAACAGCGTGTCCAGCGTCGTGGAAAGGCTTAGGC CCTACTACCGGGGGGTTGTTGTCCTTTATGGTTCTTCCTTTGGGGAGAAGTCACTCACTGGTGTTTATGACGCA AGTGACCCGGTTGGCGCATTTCGGGCGATCGCAGCCGCGCATCATGCTCAGATGCATCAGGTTTCGCCATGGCT GACAATATTGGCCGCACCGTAG SEQID6 Ferrichrome-iron ATGAAGGGTGCGGTTGCATTGCATTCGCAATTGTGGCGGCTCATACGAATGGGAACGGATAAGGTGATGACGAT receptor TGATGATAGAATGAAGCGGTGTGGGCGGCAGGTGGCGTGGCTTATCGCGCTGGGTAGTACGACGTTTCTGAATG CCGCTGTGACGAAAAGCTATGGTGCAGAACCTTCCCAAAGTGCTCGGGCCGTCAGATCATTTTCCATTCCGGCC CAATCTCTTGAPGATGGTCTCGCAAGGTTCGGACAGCAAAGTGGGTGGCAGGTTTCTGTTGACGGAAATCTTGC AAAATCTCTGACAACGCACGGTGTTAGCGGTACGATGACATCTGCTCAGGCCCTCAATGCGATCCTGTCCGGGA CTGGCCTGACATACACGATCAGGGGTGGCCGAACCGTCGTGCTGACGAAAGCAGTAGCCAACATCACGCTTGGT CCGGTCCGTGTCGGAGGAACCCTCGCGCGTCAGGATCCAACAGGGCCGGGTGTCGGCTACTTCGCCGAAAACAC AATGGTTGGTACAAAGACGGATACGCCCATCACGGAAATACCGAACTCAGTCTACGTCGTGACCAAGCAGTTGA TGACCGATCAGCAGCCGCAGAATATCCTACAGGCTTTGCGTTACACTCCCGGCATCTACTCTGAAGCCGGAGGA ACGACAAATCGCGGATCTGCCCAGAATGACAACATGGGCATTTATCAGCGTGGATTTCTCTCGAGCCAGTTCGT GGATGGGTTGATGACGAATTCGTATGCCGCCGCCGAGCCAAGCTTTCTGGACCGTATCGAGGCGCTCAACGGTC CAGCATCGGTGATGTATGGCCAGACGACACCCGGAGGAATGGTCGGTATGAGCCTGAAGAAACCCACCGAAACG CCGCTGCATCAGGTTTCGCTAGGCTTCGGAAGCTGGGGACGGTACGAGGCAACGTTCGATGTCAGCGATAAGAT CACGCAGTCCGGTAATCTGCGCTATCGTATTGCAGCCATCGGAGTCACATCGGGCACTCAGGAAGACCGAATTG ATTATCATCGGGTGGGTGTACTTCCTTCAATCACGTGGGATATCGATCCCAAGACTCGCCTCACTCTACTTGGT AGTTATATGTACACGCCTGGCTCAGGGAGCACCAGTGGTTATCCGGTCCTCGGGACTCTTATTCACAATTCGGA AATTCCACGAATCTCACGAAGCACATTTATCGGAATACCTAGTTGGAACACTATGGGAGATAAGGTCGGGATGT TCGAATATCAATTTAGTCATAAATTTAATAAATTTATTGAGTTCAATCAGACGTTTCGCGTAGAGAATTCCAAC GTTCATGAGTCAAATATCACCGATGTAACACCTGTAGATGTTGAAGGAAAATGGACATATTTTTATCCTTGGAA ACAAAATTATGAAAACACAACTGAGGTACTTGATACTCGCTTAGGGGGGCGGTTTCTAACTGGTCCTGTACAAC ATACATGGGTCATCGGTTCTGATTTCCGCAATTATGACTATCATTATACTGAGCTCATCGACGACGGTGCGACA ATCGTTGTGCCCACTCAGCACCCGACATCAAACTATTCCCCATGTATAAGTTTAACCTCCGCGAAGTGTGACGC CTTCGCGGGAATAAACCCAGACTATAACTCGTTTCAGGAGGGCGTGTATTTTCAGGACCAGATAAAATGGCAGC GCCTGTCCGTTCTCTTGGGTGGACGCCAAGATTGGGTTAATTCATCTAATAAAAATTACAGTGTAACGAACTTT TATGGAAACGTCAGCACCCGCGTTAATAACACTGCTCCACACCCTCAATCGGCCTTCACCTGGAGAGCTGGTAT AATCTATAATTTTGACTTTGGGCTTGCCCCGTACTTCAGTTACGCAACATCCTTTGTGCCACAAGGAGGTACGG ATTGGCAGGGTAAGATTTTCGCGCCTTTGAGCGGAAAGCAACTCGAAGCCGGGTTGAAGTATAAAGTTCCAAAC GAAGATATCCTCATAACGGCATCAGCATTCCGAATTGATGAAGACCACTATCTTATCAGTGATCTTGTTCACAC GGGCTTTAGCAGTGACGCGGGAACGGTACGCTCGCAGGGTTTCGAGGTTTCCGCCAGTGCGAACATTACCAAAA ACCTCAAACTTGTCGCCTCTTACACATATGAGGATGTGCGGTTCAGAAAGAACAATTTGGCCGTAAATTCGGTC GATCCCGTCACGCTAACATATGGAGCAAAGGTAAGCGAGAATGGAAAATTCGTTCCTCGAGTTCCTCGGAATAT GTTTAATATGTTTCTTGATTACACCTTCCACGACGCCCCATTGAAGGGTCTCGGCTTTAATGGAGGAATTCGCT ACACCGGTTTTACCTATGCGGACTATGTGGAGTCTTACAAAACGCCGGCGTATTATCTGTTTGACATTGGCGCA CACTATGATTTTGAGGAAATAATCCCTTCTCTCAAAGGTCTGCGTGCCCAGTTGGCAATCTCAAATTTGGCCAA TAAATATTATATTACTTCGTGCAATACCGCCATATGTACTCTCGGTTATGCTCGAAAGTTTTACGGTAACGTGA CGTATAGCTGGTGA SEQID7 Reverse GTGACGCCCGAATTGCTCCTCTCCAAGGTGCGGCTGCTGCGGTCGCCCAATGACGACGGCGCGTTCTTCGACCT transcriptase AGTCGGCAGTGTTCTTAATTGGTCCTGGGAGGAAAGAGACGAACGTCAATTCGCCCGCTTCAAGCAGCGCGCGG familyprotein GCATCCCTGAGTTCGATGGCGTCGCCCTTCCACAGGGTTTGGTTGCAGCTGGCTTCTTCTCGAACATCGTGCTG CTTGATTTCGATCGGATCGTCATCGGACAGATTGGGAGAGAAGTTACAAACGGAGTGTGGCTCCGGGACGCCTG CCGGTACGTCGACGACATTAGACTGACCATAACAACTGCACCAGGTATTGACCCAAGAGAAGCTCAGGCGCGTG TAATGGCGTGGCTTGGGCAACTCCTCACGGGGAGCTGTCCGGGCTTGGAATTCTCCCCGGAGAAGACGTCAACT GCGTCGGTTGGAGGCGAGCAGATGCCGCTGGTTCGCCAATCCCGAAAGATGGAGCGCATCCAGACCGCGATTTC CGGCGGCTTCGATGCCAGTGGTGGCGAGGAGGTGATCCACGCGATCGAAGCCCTCGTCCGATCCCAGCTAACGA TCAACAGCGTCGAAGAGTCGCCTACCCCTCCCGGCTTGAGAGCGGTACCCGATGTCAAAGACGAGACAGTCGGT CGTTTCGCTGCTGGTCGGTTCAGAAAAACCTTTCGTTCATTGAGGCCACTACTCGATGATCGACCTTACATGGA GATTGCTGAATTCGGGGAGGAGACGTTCCGGCGCACCCGACTTTCGCAATCGGAGCTTGACGAGGAAGCACGCG CATTTGCGCTAATCCTAGTCGAACGGTGGATACTCGATCCTTCGAATGTGCGGCTGCTGCGCGTCGCACTCGAC CTCTGGCCGTCCCGCCAACTCCTCAAGGAAGTACTGAAACTCTTTGAGCCCTATCTTGTCGGGAAGATCAGGGC AATCACTAGCCGGCAAGTTGCATACTACTGCCTCGCCGAGATATTTCGAGCAGGGGCGACCGAGACGGGCTTCA TTGACGATCCAGAGTGCCTTCCCGCTGCCGTCGATCTCGCCGGTTATAGATCTCTGCTTCTGGAGGCCGCAGTA CGAGTGGCCCGGGGCGAAGCCGAACGTGTCCCGTGGTATCTCGCGCAACAAGCACTGCTTTACATTGCGGTCCA CGATCCCCGGGCTATCCAAGATCGAGGAATTTCAAAGACCAATCGATCCTATTGGCGCCTCGTCTCATTTCTGA AAGGCGAACGCGACGTCTCTTCAGATCGCGAATTCGCAGTAGCCGCGGTGGTGAGCCGCAGGTCGTTCCTTTCG AATGATCAGGCCGTGGATCTCGTCGGTCGGATGCTCACGCCAGAGCGGTTCGCCGAGGTGGCCGCGCGCGACAT AGAATTCGCCCGCGATCTCTTTCGCGCCGTCGACCGACACCTCACCGTTCCGGCAGGCATTGCCGAGGACTTGG GGGTCGCCGAATGGTCCATGTCAGAGGAAATGAGCTCTCTGCAAAGCTATATCCAAGGCAAAGGGCCTCTGAAT CCGCTACGCAATGAGATCGGCGTACTCAGTTTTGCAGAGAAATTCATCTCCCATCTCCAAGAAGGAAATTTGCC GGAAGTCGTGACGCCGTCGACGACGCAGATAGCGGTACAGCAAGTGGGCAAATATGTCCGCGTCGAACGGGTGA TCTTCAGATCGGCCCAGACAACGCCGACTTACCGGTCTATTTATACTGCTCCCAGATGGGCGCCGGAATCTCAA CGCTGGCGCTTTCAGCTCGGTTATTTACTTCGCTTCATTCTTACTGCCAGAATAGACTTCAGCCTTCCAGTTAG GCCGCCATCGTGGAAGGAAGGTAAACACATCTATCGGCCTACCAGAAGTCACTGGTTTCAGCGGCAATACGGCT TCTATAATGGGCATGAGGCCTTCGGGGACGATTGGCTACCCATTTCGCAGTTCACTCAGGATCTTCTCTTCGAT CTGCTCACCTGGCCCGGCTGCCGCACAAGTAGCCCGGATGTCGATCAGTTGTCCCTGGATGAAACGGCTGCTCG AATCCGCGCAGCTCTCGTAGAAGCCACCGCTGCGATTGGCCCGGCTACAGGAACCCTGATGCTCAAGATCGATG CACCTATTCCAGGTACCACATCGAAGGGGCGCCCGCTTCGGGCCTGCGTCGTCCAGTCGATCATGCCCGAAGCA GAGGACTTCTCGGCTGCCGATCTGGAGATGCGCTCGCCGGCCCTTCGACGAAAGCACCGCAAACATCTGTCCAC GGCATTGGCGGCGGTTGAGAAGATGTTGGATCTTCGCGAGACTCATAAGCCAGCCAGCAAGCGTCTCGACTGGC TCATCCTACCAGAACTGTCTGTTCACCCGGATGACGTTGCCACCCACCTCGTGCCGTTCGCGCGAGCGTTCAAG ACCGCGATCCTGGTCGGCATGGCCTACGAACAAGTCGTCACGGGAGAGCCGCTGATCAACTCGGCCCTCTGGAT TATCCCGAGGATGGTGCGGGGCATGGGCCTACAGACGGTGATCAGACGGCAGGGAAAACAGCACCTCTCTCCGA TGGAACAGAAGTACGTCAAACCGGTCGAACTGATCACCGGATTCCGCCCGTGCCAGTGGCTGGTGGGGTACGAA TGGTCGAACAATCCGGCCAAAGACGCACTTTGGCTCACCGCGTCCATCTGCTACGATGCAACAGACCTGAAGCT GGCGAGCGATCTTCGTGATCGCTCAGACGTGTTTGCGATCCCAGCCCTGAATCTCGACGTCGGCACCTTCGATC AGATGGCGCAGGCGCTGCATTATCATATGTTCCAACTCGTGCTGATCGCGAACAACGGAGCTTATGGGGGCAGC AATGCTCACGTTCCCAAGGGGGAGGCCTATCAACGCCAAGTGTTCCATACCCATGGCCAGCCCCAGGCTACAAT TTCCTTTTTCGAGATCGACGATATCGAGGGCATGAAGCAGAGACACAAGCTCGGCGCTGGGAAGGAAGGCGGGT GGAAATATCCACCTGCCGGCTGTCAAGTCTGA SEQID8 DNAtopology TTGATGCGCTTGTTCGTGACGGGGCCAACTGGCAGTGGAAAATCAACGCTGGCTGCAAAGTTGGCTCAAAGGGC modulation AGCTATACCACTGTTCCCGCTCGATGAAATTCATTGGGTTCGCCATCTCTCCGGGGATTGGCGGCGCGATCCTG protein TTGAACGCCTGTCTATGCTCGGAGAGATTGTACAGCTCGATGCCTGGGTCATCGAAGGCGTGCAGTTCAAATGG ACTGATATAGCGATAGAACGAGCAGACTGGATCGTCGTCCTCGATCCACCACGTTGGCGGAACATCGCTCGTAT CCTGCGCCGTTTCGTCAATCGCCGATGCTTTTCTGGGGCGGGGCACCGTGGAACGGTAAAGGCTCTATTGGAGG AGATGCGTTGGTCAGCCGACTACTATGGTCATGAACGCGGTATGCTGTTCGAGAAGATTGGACAATCGCCAGAC AAGCTCATCGTCGTACATGACGACAAGGGCGAACGCGCTTTGACCGAGGCTGTATTCGCGACTGCGTGA SEQID9 Cycloisomal- ATGGCATATTGGATCAGGCTCTCGCTGGCCGTGTGGCCGCCCGATCAGCAACGTTGTAGCGAAGGCCGCGTAAT tooligosaccharide GCGCCGCTATCTTTTCACAACCATTCTCTCGCTCTTACCGTCCCTTGCGGCGGCGGCATCCCTCCAAGGTCCGA glucano- TTGTTTCGCATGTGCGGGATGATCGGGCTTTCTACCAGGCAGGCAATGTCGCGATGATTTCCGTGGAACTGACC transferase CCACTAGCCGCTTGGACGGGAGGCCATGTGGATCTAGCGATATGTTCGCGTGGGCAAGTCGTGGGCACGATTCA precursor GAGCCAAGCGGTCACCAGCATGGTGGCTGGGGCGGACCAGACACTTCACTATCCCGTCACCGTTCCCAGTCTCC (EC2.4.1.-) ATGCTCATGGGTATCAGTTGGCTATCGCGGCCCTGAACAATGGGGACAGCGGGACAGCGTCCTGTACCGGGACA GGCAGCACTTCCACGTCGCCGGCCGATGTGGCGTCAGGCGGCATCAACGTGGCCGCGAATGCCTGGGAAGACAT CAACGAAGCATGGGTCGACGCGCCGACGCTCGGCAACGTCTCCGCGGCCCGGGTGATGGATAATCTCAGCCAGT ATCACATCAACGCGGTGCAATTTTACGACGTGCTGTGGCGACATGACGAGCCGTATTCATCCGCCCCGCAGTGG CCGAACCTGGAAGGCGTAACGGTCACAAGGACCAATCTTCAGGCTTATATCAGCGCGGCGCATAGCCGCGGCAT GGTGGCGCTCGCCTATAATCTCTGGAACGGAGCCTGGGCGGACTATGCAACTGTCAATCCGAAGGTCACGGCGG CAATGGGGCTCTATGCTTCGTCCGGACAGAAACACCAACTGACCAACGGCGGGGGCTGGCTGTCCTGGGGGTGG TCGACCGACCATATTGCTGAAATGAACCCGTTCAATGGCGACTGGGCCAGATGGCTAACCAGCCAGATCCAGAA GACCATGTGGAATTTAGGATTCGACGCCGCGCATCTGGATACGTTGGGTGACCCTGGTGGTCAGCAATATGACG GCGAGGGCCATCCGTTACCTGCTTTAGGAACGATTCTGGCAGACTTTGCGAATAATGTACAGGCTCAGACCGGG GCACCAACTGACATCAACGCCGTTTCGGGTTGGAATACCACCGACCTTTACCTACGCGGTACGGGACCCAACCT GTATATCGAACCCCATCCCGAATTCGGAAACACGCCGGGCTACGATGATTCCCGAAGCTTATGGGACATCAAAC AGAAATATACGTCGCGCCCGCTGATGACGGCGTTTTATCCGCAGCAGGTCCAGAGCGGTTCGCTGAGCACGTCC TTTGCCGTCAAGGGTGAGAGTGTGAAGGTTTGCGACCCGACGTTAAAATCCGGATGCATAGCCAATAACCTCGG CATTGAGTTGTTGCTCGGCCAGATTGCGCTCAATGGAGGCTCCAATATTACTCTTGGTGATTTTGATCATCTGA TACCGGGGCCATATTTCCCCCGTCCGACCCTTAAGATCGACGGTCCATTGCAGCAATATCTGGCGGATTACTAC AACTGGTGGGTCGGAATGCGCGATCTGCTGCGTGTCGGCGTCATCTCATCCAATGAGAGGGAGTCCATCCGGAA TGGAAACGGAGCCAGTATCGGCCAACCTTATGCCCAACCGGGAACCGTCTACTATCATCCCCTGATACGCGCTG GCATCGCTGGTGAATTGGCGCTCACAAACATGATCGGGTTGCATTATAATCGGATTGACGACCCTGACGGCAAA AACAATCCGACCCCGGTGAACAACCTGTCGATCGAGATGGAATTCTGGGAAAGAAGCACACCGGGGGCATTGTA CTATAGCGCGCCCGACATCAACCACGGCTTCCCACAGCCCCTCACCTATAGGCTGAACGGAAACGGTAGCGTGA TGTTTACGCTACCGACTCTCAAGACGGTGGCGCTTGTTTGGCTGGAAGGCACCAATTTCACCACTACGACCGAT TACACGATCGGTACGGCGCAGGATGTGAGGGGTGGCACAGCAAACTTCTGGACGAACGGCAGCGGAGAGGATGC TACCGGATATCGTGGCTGCTGTGGTCGCTCCGCACGCTGGGACAGCATCGATTTCGGAGCGGGTGTGTCAACGC TAACGATGGTAACCCGAAGCCAACTCGGCGGACTGGTCGAATTTCGCCTGGATGCACCTGATGGACCAGTCATC GCCCGTAATTATGTTCCTGCGTCTAGCGCCACGACAACAACCACTCAATTACGCAGGCCAGTATTCGGGACACA TACCGTCTTCGCTAAAATTCCTGGTCGCGAGATTACGCTGATATCCTGGAAGCCATAA SEQID10 Methyltransferase ATGATGGCTAACGACAATACCACTGAGGTGGTTGGTGCATTTGCGGTAAGTCATCCCAACTTGGCGCAAGGTTT (EC2.1.1.-) TACTTTTAGTAACAGCAGTCAACTAGATACGATTGCTTCTACTATTCATAAAAGCGGTTTGGAGACTTATGAAG CTCCGACAACTAATATAATTATCGAACTGATCAGGAGTTCGTCTGGTCTTATTTTAGATGTGGGAGCGAATACC GGTCTTTTTACGCTAGTCGCCGCAGCAGCCAACCCCCTGATCCGCGTCTGCTCTTTTGAGCCGCTTGCGAGTAT TCGTGAACTTCTCAAGAGCAATATTGCTCTCAATCCGGAGCTTGCATCACGTATCGCTGTCGAGCCTGTCGGGT TATCGAATGAACGGGGCACTTTCACTTTTTACGAAACGATCAACAATCGTGGCTTTGTCACGACGAGTTCATCG CTTGAAAAAGCACATGCAGAGCGAATCGGCGATTTGTACGTCGAGCGCACTATCGAGACCCGGACACTTGATGA ATTCGGAGAAACGCTCGGGAATGCGAGCGTTCCGTTCGTCAAAATTGACGTTGAGGGACATGAGCATGCCGTTA TCTCCGGTGGCCGCCACTTTATCGCCAAGCACCGCCCTTTTCTTACTCTCGAAGTCCTGAGAGAGGCTAACACT TTGAGTCTGGACCAGTTGGTGACCGAGTCCAACTACCTTGCCCTGGCAATGGCACCCGACGAATTGCGGCAGTG CGAGCGTTTACGGTTTCATGACGACGCCTGGAATCATCTTTTGGTCCCCGCCGAAAAAGCGGAACGGCTATTTT CGCTCTGCCGCCGACTTGGCTTGCAAATCGGGATCCGCTGA

(75) TABLE-US-00007 TABLE2 SEQID11 Transcriptional ATGTCGAATTCCGAGCGCCCAATGCGCGATTTGTCGGACCTGGCAAAAAACCGACAAATAGAGCCGATGGTTAT repressor; CAGGCTACGAGAAGTAGTGGATCGGACCGGAGGCGCGAAAGCTGTGGCCGCACGCACGGACATCCCTCTCAGCA asserted_pathway: CACTTTCAGGTTACCTGTCGGGTCGGGAACTCAAGCTTTCCGTCGCGCGCAAGATCACGGAAGCCTGCGGTGTC PF01381<br> AGTCTTGACTGGCTTGCGGCAGGAGAGGACGGACCTGCGGCCCGGGAATTCGGCAATGCCCGGCAGGCGGGTCC PeptidaseS24- CGAGTCGGTCGAGTTTCTGAATTACGACGTCATTCTCTCCGCCCACCAGGGCGTCGACGGGGATAGTTCTTATA like::PF00717 TCGAAACGAGAATATCGATACCGCGGGATTTTCTCCCTTTGTCCATTCAGTCCAATACGGACAACATTTCGGCC GTCACGGCGAAATGCGACAGCATGAATCCGATCATAGACGATGGAGACATTCTTTTAATAAGAACGGATGTGCA TACGCTCACAAGTGGCAGCATCTATGCCCTGCGGGTAGAAAACACCCTTCTGGTCAGGCGTCTGATCCTCAAGA CCAACGGCAACGTCCAGGTCATCAGCGAGAACCCGCGTTACCCGACCGAGGAACTGAACGCCGAGGACGTTCGC AGGATGGTCCAGGACGACGGCTTTCCGGCCAGGATCATCGGCCGGGTCATCTGGCGCGCCGGTAGCCTGATTCC ATAG SEQID12 outermembrane ATGCGCATCGTCCTCTTGCCCTGCCTCGTCGCGACCTCAATAAGTATGTTGGCGGTTTCCGCATCCTATGCTTG hemereceptor GGCGGACAATAGCCCGTCGCCCCCCAGGACGAACAAACAGGCCAAATCGCGGCCGTTACATGCGCAGGGGACGC GCAAAGCGGGCAGCGCCATCACCAGCCAGGATGAAGCGGTGGCTGTCGTGGGAACACGTGAGACATCGCATGGG ATGGAGCAGAGCGTTACCCGTGCGACGATGGACAAGTTCGTGGCGGGGACCAGTCCCCTGCAGATTCTGTCGGC CACGACACCGGGTGTCAATTTTGCCTCGGACGACCCGTTCGGCCTGGATACATGGGCGAACACATTTTATATTC GCGGCTATTCCCAAAGCCAGTTGGGCATCACCCTGGACGGTATCCCGCTGGGCGATGCCCAGTTCATCAATTCC AACGGCCTCGATATCAATCAGGCGATCATCCAGAACAATATCGGTCGCGTCGACATGTCGCAGGGTGGCGGTGC GCTCGATGTCATGTCCGTCACCAACCTGGGTGGCGCGCTGCAATATTATTCACTCGATCCGCGCGACAAGGCTG GTGGAGACATTTCACAGACGTTCGGCAGCAACCAGACCTATCGCACGTACGTCAGCGCCCAGAGCGGCAAGCTC AATCCCAGCGGGACGAAGTTCTATGCGTCGTACGCGCGCACCGATGCCGGGAAATGGAAAGGCGCCGGGGACCA GTTCGAACAGCAGGCGAATTTCAAGATCGTACAGCCGCTCGGGCGTTACGGAAAACTGTCCGGATTCTTCAATT ATTCCGAATTCGACCAGTATAATTACAGCGATTTGAGCCTGGAAATCATCCAGAAGCTCGGCCGGAACGTGGAT TATTTCTATCCGAACTACAAAGCCGCGTATCAGGCTGCCGAGGGGATCTATCCCGCAGGCTATGCCAAGGTCGG AGATGCCATGGACGTCTCCTATTACGATGGTGGCCAGGACCAGCGGAATTATCTTTCCGGCATCACGTCCACGA TCGACCTGACGTCCCGCCTGCATCTGAAGACGGTGCTGTACGACCAGCAATCGGCGGGGGACTACGAATGGACC AACCCCTATGTGTCGTCGCCCTCGGGCGCGCCCATGATCCAGCAGGTCGGGCACACATCGATGACGCGCGTGGG CGGGATTGGCGCGGTGCAGTACCAGATCGCCAATCATTCGCTTGAAACCGGCGTCTGGTACGAAAACAACGGAT ATAGCTGGGCGCAACGGTACTACAACCAGCCGCTTCTGGGGGAGGGTACGCCCCGAAGCGCCACCGGACCGTAC AACGATCCGTTCGCCACCGCATACGCCATGACCTTCAATACCAACAGTTTTCAATATTACCTGGAAGATTCCTA CCGTATCTTGAAGACGCTGCGGGTGCACGCGGGCTTCAAATCCATGCTGACGACGACGTCGGGCGGCGCATCCT ATAACAATCCCGTCTATACGGGCCAGGACACCCTGCCCAGTGGCAGCCTGACCACCGCCAGCGCCTTCCTGCCG CATGTCAGCATCAACTGGAATTTCCTGCCCCGGAACGAACTGTTTTTCGACTTCGCGGAGAACATGCGCGCATT CACCTATAATACATGGCAGAGCGGGAATGCATGGGGAGTCAATGAGATGCCCCAGAACCTGAAGCCCGAGACCA CCTTCAATTACGAGGTCGGTTATCGATATAATTCCCGCTTCGTCACGGGCCTCGTCAATCTGTATCATATCGAT TACAGGAACCGGCTGGCCACCATCACCACCGGCAGCCTGGTGAACGCCCACAATACCTATATCAACGTGGGGAA CATGGCGATCTGGGGTGCCGATGCCGGCGTGACGGTGCGCCCGCTGCCGGGCCTCGAGATCTTCAACAGCGCCA GCTACAACAAATCCACCTATGGGCAGGATGTATCCAGCGGCGGGGTAAATTATCCCATCAGCGGCAAGCAGGAG GCCGGCTATCCGCAATGGATGTACAAGGCCAACGTCTCGTACAGGTATGGCAACGCGAAGGTCAACTTCAACGT CAACTATATGGGAAAGCGATACATCTCGTACATGAACGACGCCGCCGTGAACGGGTATTGGCTGGCATCGCTGT CGGCGACGTATATCTTCAAAACCATTCCCCATCTCTCTCAGCTTGAATTCAATTTCGGCGTCTACAACCTGTTC AACCAGGAATATATCGGCGGCATCGGCGGGTTCTCACTGTCCGGTGACACGCAGCAACTCTTTGCCGGCGCGCC ACGCCAGTTCTTCGGTACGCTGCACGCACGGTTCTAG SEQID13 Levansucrase GTGACGGCGCGGTCGTGGTTGCTCTGCAATCTGAAGAGTTTCCTTCAGGAGGATGGAATGGCGCATGTACGCCG AAAAGTAGCCACGCTGAATATGGCGTTGGCCGGGTCCCTGCTCATGGTGCTGGGCGCGCAAAGTGCGCTGGCGC AAGGGAATTTCAGCCGGCAGGAAGCCGCGCGCATGGCGCACCGTCCGGGTGTGATGCCTCGTGGCGGCCCGCTC TTCCCCGGGCGGTCGCTGGCCGGGGTGCCGGGCTTCCCGCTGCCCAGCATTCATACGCAGCAGGCGTATGACCC GCAGTCGGACTTTACCGCCCGCTGGACACGTGCCGACGCATTGCAGATCAAGGCGCATTCGGATGCGACGGTCG CGGCCGGGCAGAATTCCCTGCCGGCGCAACTGACCATGCCGAACATCCCGGCGGACTTCCCGGTGATCAATCCG GATGTCTGGGTCTGGGATACCTGGACCCTGATCGACAAGCACGCCGATCAGTTCAGCTATAACGGCTGGGAAGT CATTTTCTGCCTGACGGCCGACCCCAATGCCGGATACGGTTTCGACGACCGCCACGTGCATGCCCGCATCGGCT TCTTCTATCGTCGCGCGGGTATTCCCGCCAGCCGGCGGCCGGTGAATGGCGGCTGGACCTATGGCGGCCATCTC TTCCCCGACGGAGCCAGCGCGCAGGTCTACGCCGGCCAGACCTACACGAACCAGGCGGAATGGTCCGGTTCGTC GCGTCTGATGCAGATACATGGCAATACCGTATCGGTCTTCTATACCGACGTGGCGTTCAACCGTGACGCCAACG CCAACAACATCACCCCGCCGCAGGCCATCATCACCCAGACCCTGGGGCGGATCCACGCCGACTTCAACCATGTC TGGTTCACGGGCTTCACCGCCCACACGCCGCTGCTGCAGCCCGACGGCGTGCTGTATCAGAACGGTGCGCAGAA CGAATTCTTCAATTTCCGCGATCCGTTCACCTTCGAGGACCCGAAGCATCCCGGCGTGAACTACATGGTGTTCG AGGGCAATACCGCGGGCCAGCGTGGCGTCGCCAACTGCACCGAGGCCGATCTGGGCTTCCGCCCGAACGATCCC AATGCGGAAACCCTGCAGGAAGTCCTGGATAGCGGGGCCTATTACCAGAAGGCCAATATCGGCCTGGCCATCGC CACGGATTCGACCCTGTCGAAATGGAAGTTCCTGTCGCCGCTGATTTCGGCCAACTGCGTCAATGACCAGACCG AACGGCCGCAGGTGTACCTCCATAACGGAAAATACTATATCTTCACCATCAGCCACCGCACGACCTTCGCGGCC GGTGTCGATGGACCGGACGGCGTCTACGGCTTCGTGGGTGACGGCATCCGCAGTGACTTCCAGCCGATGAACTA TGGCAGCGGCCTGACGATGGGCAATCCGACCGACCTCAACACGGCGGCAGGCACGGATTTCGATCCCAGCCCGG ACCAGAACCCGCGGGCCTTCCAGTCCTATTCGCACTACGTCATGCCGGGGGGACTGGTTGAATCGTTCATCGAC ACGGTGGAAAACCGTCGCGGGGGTACCCTGGCGCCCACGGTCCGGGTGCGCATCGCCCAGAACGCGTCCGCGGT CGACCTGCGGTACGGCAATGGCGGCCTGGGCGGCTATGGCGATATTCCGGCCAACCGCGCGGACGTGAACATCG CCGGCTTCATCCAGGATCTGTTCGGCCAGCCCACGTCGGGTCTGGCGGCGCAGGCGTCCACCAACAATGCCCAG GTGCTGGCGCAGGTTCGCCAATTCCTGAACCAGTAA