PRIOR ART Substituted phenylurea herbicides are used to control weeds in a wide range of crops and in amenity horticulture. However, detection of these compounds in drinking water supplies has led to restrictions in their use.

Although some bacterial and fungal isolates have been reported that are able to breakdown some of these compounds, most strains have not been well characterised (34). Even amongst strains which have been characterised, no genes involved in the breakdown of substituted phenylurea herbicides have been described.

DISCLOSURE OF THE INVENTION The present inventors have succeeded in identifying and isolating a novel sequence encoding a phenylurea hydrolase from Arthrobacter globiformis D47 which is responsible for herbicide degradation.

Strain D47 was originally isolated from a soil that had developed the ability to rapidly degrade the phenylurea herbicide Diuron, which is usually relatively persistent in soil (7). This strain was also to degrade the herbicides isoproturon, chlortoluron, linuron and monolinuron. Degradation of all these compounds was shown to occur through hydrolysis of the urea carbonyl group (7). Subsequent characterisation of the strain D47 indicated that it belonged within the Arthrobacter globiformis group (36).

Specifically, the present inventors have isolated a fragment of a plasmid found in Arthrobacter globiformis D47, and shown that this fragment confers phenylurea hydrolase activity on E. coli.

Within this fragment the inventors have identified two open reading frames, one in the forward strand (called ORF2 herein) and one in the reverse strand (called ORF4 herein).

The isolation of the sequence responsible for phenylurea hydrolase activity was achieved notwithstanding a number of complications. For instance, distinguishing between degradative. and non-degradative isolates hampered by the fact that the degradative phenotype appears to be unstable. Moreover, the degradative phenotype did not appear to offer a selective advantage to the degradative strain, presumably because carbon-sources in the media other than Diuron were being metabolised for primary growth. Only a small proportion of the colonies on the selective media in fact degraded Diuron.

Because of the presence of ribosome binding sites on the reverse strand, it is believed that the phenylurea hydrolase activity is encoded by ORF4. However, it will be appreciated that the various aspects of the invention discussed below apply correspondingly to any other ORF (e. g. ORF2) within the disclosed sequences which encodes the relevant activity. The appropriate ORF may be verified by the person skilled if it is desired to do so as follows: PCR primers may be designed to selectively amplify one ORF. This amplified ORF may then be cloned into an expression vector and expressed in a suitable host. The enzyme activity of the expressed clone may then be determined using techniques as described herein.

In a first aspect of the present invention there is disclosed an isolated nucleic acid molecule comprising a nucleotide sequence which

encodes a phenylurea hydrolase obtainable from a Coryneform bacteria such as Arthrobacter, preferably A. globiformis, most preferably A. globiformis D47.

The nucleic acid and other embodiments of the invention described below have utility, inter alia, in methods of bioremediation, biocatalysis and detection of phenylurea herbicides.

Unless context demands otherwise, where the term phenylurea'is used herein it should be construed broadly and taken to cover not just phenylurea but also derivatives thereof and similar compounds.

Examples include those having the following formula: Where Ri is CH3 ; R2 is CH3 or OCH3 ; R3 is H or Cl ; R4 is Cl or OCH3 or CH(CH3) 2.

Phenylurea hydrolase activity'means the ability to hydrolyse a phenylurea, for instance to convert phenylurea into aniline and carbamic acid. Where the phenylurea is a derivative as explained above, then the products of the hydrolysis reaction will be derivatives of aniline and carbamic acid, as would be understood by the person skilled in the art.

Phenylurea hydrolase activity may be assessed by measuring the decrease of phenylurea or the increase in the products of the hydrolysis reaction by HPLC, or other chemical methods, as described herein. Any suitable technique may be used for measuring the phenylurea hydrolase activity as is understood by the person skilled in the art.

Changes in another molecule which is involved in the hydrolase reaction may be monitored to determine the activity of the phenylurea hydrolase. For example, it is known to monitor the change in ATP levels in the case of enzyme reactions which involve the conversion of ATP to ADP.

Alternatively, changes in the phenylurea hydrolase protein structure due to substrate binding may be monitored. Or, phenylurea hydrolase fused with a suitable tag and the binding of the tagged enzyme to an immobilised substrate may be monitored.

In one embodiment of the invention, the sequence encodes the phenylurea hydrolase amino acid sequence shown in Annex (II).

In a further embodiment of the first aspect of the invention, the nucleotide sequence is that shown in Annex (I), or within the reverse strand of the nucleotide sequence shown in Annex (I). This embodiment embraces the nucleic acid sequence from nucleotide 2298 to 928 of the complementary strand of the sequence shown in Annex I (ORF4').

Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term"isolated"encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e. g. using an automated synthesiser. Nucleic acid according to the present invention may include cDNA, RNA and modified nucleic acids or nucleic acid analogs.

Where a DNA sequence is specified, e. g. with reference to a figure,

unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Where a nucleic acid (or nucleotide sequence) of the invention is referred to herein, the complement of that nucleic acid (or nucleotide sequence) will also be embraced by the invention. The'complement'in each case is the same length as the reference, but is 100% complementary thereto whereby by each nucleotide is capable of base pairing with its counterpart i. e. G to C, and A to T or U.

Nucleic acids of the first aspect may be advantageously utilised, inter alia, in hosts to enable bioremediation of the environment surrounding the bacteria.

In another aspect of the present invention there are disclosed nucleic acids which are variants of the sequences of the first aspect.

A variant nucleic acid molecule shares homology with, or is identical to, all or part of the coding sequence discussed above. Generally, variants may encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which are capable of hydrolysing phenylurea hydrolase and/or which will specifically bind to an antibody raised against the polypeptide of Annex (II).

The phenylurea hydrolase activity may be assessed as described above.

Variants of the present invention can be artificial nucleic acids (i. e. containing sequences which have not originated naturally) which can be prepared by the skilled person in the light of the present disclosure. Alternatively they may be novel, naturally occurring, nucleic acids, which may be isolatable using the sequences of the present invention.

Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided. The fragments may encode particular functional parts of the polypeptide.

Equally the fragments may have utility in probing for, or amplifying, the sequence provided or closely related ones. Suitable lengths of fragment, and conditions, for such processes are discussed in more detail below.

Also included are nucleic acids which have been extended at the 3'or 5'terminus.

Sequence variants which occur naturally may include alleles or other homologues (which may include polymorphisms or mutations at one or more bases).

Artificial variants (derivatives) may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid. is generated either directly or indirectly (e. g. via one or amplification or replication steps) from an original nucleic acid having all or part of the sequences of the first aspect. Preferably it encodes a phenylurea hydrolase.

The term variant'nucleic acid as used herein encompasses all of these possibilities.

Some of the aspects of the present invention relating to variants will now be discussed in more detail.

Homology (i. e. similarity or identity) may be as defined using sequence comparisons are made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap):-12 for proteins/-16 for

DNA; Gapext (penalty for additional residues in a gap):-2 for proteins/-4 for DNA; KTUP word length: 2 for proteins/6 for DNA.

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology with Arthrobacter globiformis D47 phenylurea hydrolase.

Thus a variant polypeptide in accordance with the present invention may include within the sequence shown in Annex II, a single amino acid or 2,3,4,5,6,7,8, or 9 changes, about 10,15,20,30,40 or 50 changes, or greater than about 50,60,70,80,90,100,200 changes. In addition to one or more changes within the amino acid sequence shown, a variant polypeptide may include additional amino acids at the C-terminus and/or N-terminus. Naturally, regarding nucleic acid variants, changes to the nucleic acid which make no difference to the encoded polypeptide (i. e. degeneratively equivalent') are included within the scope of the present invention.

Thus in a further aspect of the invention there is disclosed a method of producing a derivative nucleic acid comprising the step of modifying the coding sequence of a nucleic acid of running from nucleotide 2298-928 of the complementary strand of the nucleotide sequence shown in Annex I.

Changes to a sequence, to produce a derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage ; other sites which are required

for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide (e. g. binding sites).

Leader or other targeting sequences (e. g. hydrophobic anchoring regions) may be added or removed from the expressed protein to determine its location following expression. All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form (as described below).

Other desirable mutations may be random or site directed mutagenesis in order to alter the activity (e. g. specificity) or stability of the encoded polypeptide.

Changes may be by way of conservative variation, i. e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure.

In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those

described above may confer slightly advantageous properties on the peptide e. g. altered stability or'specificity.

In a further aspect of the present invention there is provided a method of identifying and/or cloning a nucleic acid variant from bacteria which method employs the sequence running from nucleotide 2298 to 928 of the complementary strand of the sequence shown in Annex I, or a derivative thereof (e. g., fragment, or complementary sequence. Target bacteria include (but are not limited to) those of the Coryneform bacteria, especially Arthobacter species.

An oligonucleotide for use in probing or amplification reactions comprise or consist of a distinctive sequence of about 48,36 or fewer nucleotides in length (e. g. 18,21 or 24). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-30 nucleotides in length (which sequence is not present in genes of the prior art) may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or more nucleotides in length.

In one embodiment, a variant in accordance with the present invention is also obtainable by means of a method which includes: (a) providing a preparation of nucleic acid, e. g. from bacterial cells, (b) providing a nucleic acid molecule which is a probe as described above, (c) (c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.

Test nucleic acid may be provided from a bacterial cell as total DNA (plasmid and chromosomal), plasmid DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector.

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. For instance, screening may initially be carried out under conditions, which comprise a'temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (e. g. Standard Saline Citrate (SSC') = 0.15 M sodium chloride ; 0.15 M sodium citrate; pH 7) concentration.

Alternatively, a temperature of about 50°C or less and a high salt (e. g. SSPE'0. 180 mM sodium chloride ; 9 mM disodium hydrogen phosphate ; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology (similarity, identity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid. Suitable conditions include, e. g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42EC in 0.25M Na2HP04, pH

7.2,6.5% SDS, 10% dextran sulfate and a final wash at 55EC in 0. 1X SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na2HPO4, pH 7.2,6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. 1X SSC, 0.1% SDS. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e. g. cDNA libraries representative of expressed sequences, may be searched. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): Tm = 81.5°C + 16.6Log [Na+] + 0. 41 (% G+C)-0.63 (% formamide)-600/#bp in duplex. As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57°C. The Tm of a DNA duplex decreases by 1-1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.

Binding of a probe to target nucleic acid (e. g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below) or RN'ase cleavage.

The identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve

one or more steps of PCR or amplification of a vector in a suitable host.

Thus one embodiment of this aspect of the present invention is nucleic acid including or consisting essentially of a sequence of nucleotides complementary to a nucleotide sequence hybridisable with any encoding sequence provided herein. Another way of looking at this would be for nucleic acid according to this aspect to be hybridisable with a nucleotide sequence complementary to any encoding sequence provided herein. Of course, DNA is generally double- stranded and blotting techniques such as Southern hybridisation are often performed following separation of the strands without a distinction being drawn between which of the strands is hybridising.

Preferably the hybridisable nucleic acid or its complement encode a product able to influence a herbicide degradative characteristic of a plant or a bacterium.

In a further embodiment, hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e. g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR). PCR requires the use of two primers to amplify target nucleic acid, so preferably two primers as described above are employed. Using RACE PCR, one'random'may be used (see"PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)).

Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may be carried out as described above, but using a pair of nucleic acid molecule primers useful in (i. e. suitable for) PCR, at least one of which is a primer of the present invention as described above.

In each case above, if need be, clones or fragments identified in the search can be extended. For instance if it is suspected that they are

incomplete, the original DNA source (e. g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e. g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.

The methods described above may also be used to determine the presence of one of the nucleotide sequences of the present invention within the genetic context of an individual bacteria or within a plant, optionally a transgenic plant or transformed bacterium.

As used hereinafter, unless the context demands otherwise, the term phenylurea hydrolase nucleic acid'is intended to cover any of the nucleic acids of the invention described above, including functional variants.

In one aspect of the present invention, the phenylurea hydrolase nucleic acid described above is in the form of a recombinant and preferably replicable vector.

Vector'is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e. g. autonomous replicating plasmid with an origin of replication). Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e. g. higher plant, mammalian, yeast or fungal cells).

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene

expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences (see below), terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, signal sequences and other sequences as appropriate. For further details see, for example, Molecular Cloning : a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press (or later editions of this work). Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above discussion in respect of variants), sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

Two types of vector are of particular interest in the present context: nucleic acid constructs which operate as microbial, e. g., bacterial vectors; and nucleic acid molecules that operate as plant vectors. Such vectors may be transformed into a suitable host cell to provide for expression of a peptide of the invention. However, a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

Nevertheless, preferably, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e. g. bacterial, or plant cell. The vector may be a bi- functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the

control of an appropriate promoter or other regulatory elements for expression in the host cell.

By"promoter"is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i. e. in the 3'direction on the sense strand of double-stranded DNA)."Operably linked"means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation"of the promoter.

Thus this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention.

In one embodiment of this aspect of the present invention, there is provided a gene construct, preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention. The term"inducible"as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on"or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.

Preferred bacterial vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more

selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. Bacterial or other microbial vectors may include a signal sequence to direct the protein so that it is expressed on the cell surface, or is secreted from the cell. Examples of such signal sequences include: outer membrane proteins, for example the OmpA signal peptide; exotoxins, for example exotoxin A from P. aeruginosa.

Further examples are described in Morganti et al. (1996) Biotechnology and Applied Biochemistry 23,1,67-75; Dunn et al (1996) Immunotechnology, 2: 3 229-240 ; Morganti et al (1998) Biotechnology and Applied Biochemistry 27,1,63-70; Molina et al., Gene 116: 2, 129-138.

Regarding plant vectors, specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148). For example, suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S). Other examples are disclosed at pg 120 of Lindsey & Jones (1989)'Plant Biotechnology in Agriculture'Pub. OU Press, Milton Keynes, UK.

The promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180. It may be desirable to use a strong constitutive promoter such as the ubiquitin promoter, particularly in monocots.

If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as

resistance to antibiotics or herbicides (e. g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

The present invention also provides methods comprising introduction of the constructs discussed above into a host cell, especially a plant cell or a microbial cell, particularly a bacterial cell.

In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a plant or a microbial cell.

The term"heterologous"is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (a phenylurea hydrolase gene) have been introduced into said cells of the bacteria, or plant or an ancestor thereof, using genetic engineering, i. e. by human intervention.

A heterologous gene may replace an endogenous equivalent gene, i. e. one which normally performs the same or a similar function, or the inserted sequence may be additional to an endogenous gene or other sequence.

The host cell is preferably transformed or transfected by the construct, which is to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e. g. with respect to herbicide resistance.

Nucleic acid can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12 (22) 8711-87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green

et al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e. g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e. g. Kindle, PNAS U. S. A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has also been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e. g. Hiei et al. (1994) The Plant Journal 6,271-282)).

Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP- A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).

Thus a further aspect of the present invention provides a method of transforming a host cell involving introduction of a construct as described above into the cell and causing or allowing recombination between the vector and the cell genome to introduce a nucleic acid according to the present invention into the genome.

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention especially a plant or a microbial cell. In the cell the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.

Transformed recombinant bacteria (e. g., E. coli) over-expressing the enzyme may be useful sources of phenylurea hydrolases for the chemical industry e. g. for use in the preparation of herbicides. In such a situation it is preferred that the phenylurea hydrolase is expressed on the surface of the bacteria, or is secreted by the bacteria.

Suitable bacteria in which the gene could be expressed for bioremediation include: Pseudomonas species, Bacillus species, Streptomyces species, Burkholderia species, Arthrobacter species, E. coli, Rhizosphere competent bacteria.

Regarding plant host cells, generally speaking, following transformation, a plant may be regenerated, e. g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989. The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162. ; Vasil, et al. (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702). It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Plants which include a transformed plant cell according to the invention are also provided.

In addition to the regenerated plant, the present invention embraces all of the following : a clone of such a plant, selfed or hybrid progeny and descendants (e. g. F1 and F2 descendants) and any part of any of these. The invention also provides parts of such plants e. g. any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on, or which may be a commodity per se e. g. grain.

A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders'Rights.

A plant expressing the nucleic acid of the first aspect may be used to degrade phenylurea herbicides when applied to them, and so be resistant to that herbicide. Plants over expressing the enzyme may also be useful sources of phenylurea hydrolases for the chemical industry e. g. for use in the preparation of bioremediation agents to degrade herbicides. Plants in which it may be desirable in principle to express, or over express, nucleic acids of the present invention may include: barley, bean (phaseolus), pea, sugar beet, maize; oat; solanum (e. g. potato) ; allium (e. g. garlic, onion and leek); asparagus; tea; peanut; spinach; cucurbitaceae; yam ; rice ; rye; sorghum ; soyabean ; spruce; strawberry; sugarcane; sunflower; tomato; wheat.

For the bioremediation of contaminated land or water a weed or non- crop plant which grows under harsh conditions, or an algae may be used. Examples include: Typha latifolia, Kochia scoparia, Canna hybrida (Yellow King Humbert), Poplar cuttings, Willow, Thlapsi caerulescens, Arabidopsis halleri, Alyssum murula, Salix viminalis.

The present invention also encompasses the expression product of the phenylurea hydrolase nucleic acid sequences disclosed above-

particularly functional phenylurea hydrolase-plus also methods of making the expression product by expression from encoding nucleic acid therefor under suitable conditions, which may be in suitable host cells.

Thus, in a further aspect the invention provides a process for preparing peptides according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the peptides, and recovering the expressed peptides. The method may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.

Preferred polypeptides include the amino acid sequence shown in Annex II. However, a polypeptide according to the present invention may be a variant (allele, fragment, derivative, mutant or homologue etc.) of the polypeptide as shown in Annex II. The allele, variant, fragment, derivative, mutant or homologue may have substantially the phenylurea hydrolase function of the amino acid sequence shown in Annex II.

Also encompassed by the present invention are polypeptides which although clearly related to a functional phenylurea hydrolase polypeptide (e. g. they are immunologically cross reactive with the Arthrobacter globiformis phenylurea hydrolase polypeptide, or they have characteristic sequence motifs in common with the phenylurea hydrolase) no longer have phenylurea hydrolase function.

Following expression, the recombinant product may, if required, be isolated from the expression system. Generally however the polypeptides of the present invention will be used in vivo (in particular in planta or in bacteria).

Purified phenylurea hydrolase or variant protein, produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art.

Methods of producing antibodies include immunising a mammal (e. g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal. As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e. g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see W092/01047.

Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes. Thus, the present invention provides a method of identifying or isolating a polypeptide with phenylurea hydrolase function (in accordance with embodiments disclosed herein), including screening candidate peptides or polypeptides with a polypeptide including the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind an Arthrobacter globiformis phenylurea hydrolase peptide, polypeptide or fragment, variant or variant thereof or preferably has binding specificity for such a peptide or polypeptide, such as having an amino acid sequence identified herein. Specific binding members such as antibodies and polypeptides including antigen binding domains of antibodies that bind and are preferably specific for a phenylurea

hydrolase peptide or polypeptide or mutant, variant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.

Candidate peptides or polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source.

In a further aspect of the invention, there is disclosed use of the materials described above (e. g. nucleic acids, vectors, cells, plants, antibodies, and so forth) for the detection or degradation of phenylurea (or derivative) herbicides. Such methods may be used e. g. for bioremediation.

For example, recombinant phenylurea hydrolase polypeptide may be used e. g. to degrade phenylurea herbicides. Phenylurea hydrolase polypeptide may also be used to detect phenylurea herbicides in samples.

A method of detecting phenylurea in a sample according to the invention may comprise: (i) contacting a sample with a material as described above which provides phenylurea hydrolase activity; (ii) determining an increase in phenylurea degradation products (e. g. aniline or carbamate.) Various methods of the invention may comprise a step of contacting a transformed bacteria expressing a heterologous nucleic acid of the first aspect with a material in which the reduction of phenylurea herbicide is desirable e. g. soil. Where the polypeptide is expressed on the surface of a bacteria, or is secreted from the bacteria, then such bacteria may be used in a method of detecting phenylurea in a sample.

The invention further provides a method of influencing or affecting the nature or degree of the phenylurea hydrolase activity (and thereby the herbicide resistance) in a plant, the method including the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above within the cells of the plant. Use of the nucleic acids disclosed herein as selectable marker genes is also encompassed.

The invention will now be further described with reference to the following non-limiting Examples and Annexes. Other embodiments of the invention will occur to those skilled in the art in the light of these.

Figures, Tables and Sequence Annex Figure 1 shows a map of pHRIM620 indicating the position of the E. coli restriction enzyme sites, and the sub-clones used (A-E).

Figure 2 shows a map of plasmid (A) (pHRIM622) indicating the position of transposon insertions (B) that inactivated the degradative ability (arrow above the line), and those where insertion dis n ot affect activity (arrow below the line), and active subclones (C, D, E) using enzymes NotI (C) and SstI (D, E). The fragment (E) was cloned into pUC18. The sizes are shown in base pairs.

Table 1 shows the growth of A. globiformans D47 strains on LB and minimal media with Diuron.

Table 2 shows the sequence similarity of phenylurea hydrolase (puhA) to proteins on SWISSPROT. % identity and length of comparison (overlap) are presented, sequences are ordered as they are listed in the FASTA 6.0 table, which takes into account other factors not presented.

Annex I shows the DNA Sequence of SstI fragment, containing the phenylurea hydrolase gene (s).

Annex II shows the amino acid sequence corresponding to open reading frame ORF4 of the SstI fragment. ORF 4 runs from nucleotide 2298 to nucleotide 928 of the reverse strand of the sequence shown in Annex I Annex III shows the amino acid sequence corresponding to open reading frame ORF2 of the SstI fragment. ORF2 runs from nucleotide 919 to 2394 of the forward strand of the sequence shown in Annex I.

EXAMPLES Example 1-Plasmid profile of A. globiformis D47 and mutants Two plasmids were observed in A. globiformis D47.

In a screen of single colonies at the end of repeated sub-culture of the wild type strain, those that maintained the ability to degrade Diuron either had both plasmids or the larger of the two plasmids, pHRIM620. Isolates that could no longer degrade either had no detectable plasmids or the smaller of the two plasmids, pHRIM621.

These results indicated that the larger plasmid gave the wild type strain the ability to degrade Diuron. Restriction analysis of plasmid DNA of strains with both plasmids, and ones with either of the other plasmids indicated that pHRIM620 and pHRIM621 were approximately 47kb and 35 kb, respectively.

Example 2-Growth of mutants on media with Diuron Pesticide degrading genes present in soil bacteria have been shown to reside on plasmids, a common feature for many degradative functions (15,23,29). However, isolation and maintenance of strains that

utilise a pesticide as a sole carbon source can often be difficult, and many strains grow on minimal media that can not degrade the pesticide or have lost the ability to degrade it.

The selective nature of the isolation media originally used to obtain the wild type degradative strain (7) from soil was investigated using selected strains that only differed in their plasmid composition.

The growth of a non-degrading strain was compared to that of the degrading strain on LB agar and MSM agar containing Diuron.

On LB agar, Diuron inhibited the growth of the wild type strain at concentrations in excess of 20 jig ml-1. The non-degrader was also inhibited at this concentration, therefore the ability to degrade Diuron did not offer the wild type strain a selective advantage.

On minimal media both strains grew as well as each other, but overall showed very poor growth. Since the non-degrader cannot utilize Diuron this indicated that other carbon sources within the media had been metabolised for primary growth.

Since a key step in isolation of degradative strains is the formation of colonies on minimal media, only bacteria capable of degrading the herbicide should grow. However, out of 32 colonies selected on MSM, only 5 degraded Diuron and were subsequently characterised as the same organism. Many workers report loss of degradative ability on sub-culture of strains, even when minimal media is used.

If the degradative phenotype is unstable, constant selection to maintain it in the population is required, which again relies on the media allowing only the growth of degradative isolates. During enrichment culture, the transfer of soil into MSM would render the system non-minimal as a media, and sub-culture would also transfer dead bacteria which could act as an alternative carbon source. In addition, impurities in the agar may have provided alternatives to

Diuron, which may explain why only 16% of the isolates showed Diuron degradation (Cullington and Walker (1999) Soil Bio. and Biochem. 31, 677-686). Many other studies report isolation of non-degrading bacteria on media specifically designed to select only for isolates capable of degradation, and in many cases the pesticide is probably not acting as the sole carbon source. In most studies, broth culture appears to provide the most stable conditions for maintaining degradation and suggests that the addition of agar to the media may also introduce additional nutrient sources.

A. globiformis D47 was unable to grow in MSM liquid media with Diuron, although degradation of Diuron could be detected indicating that the enzyme within the cells was active. However, the liquid enrichment culture must offer some selective advantage for the growth of strains to have enabled the original isolation of A. globiformis D47 from the complex microbial population in soil.

Example 3-Cloning of the degradative gene Digestion of pHRIM620 with EcoRI gave two large bands (17 and 22 Kb) and one smaller band (2.5 Kb), while digestion with SacI gave numerous smaller bands. Plasmid DNA from A. globiformis D47 was cloned into supercos and pUC19. To select clones with inserts from pHRIM620, a series of unique clones were used as hybridisation probes and screened against strains with both plasmids, pHRIM620 alone, pHRIM621 alone and DNA from a plasmid-free strain. Clones that hybridised to all samples were believed to have inserts that originated from chromosomal DNA, and only those that hybridised with strains with both plasmids or strains with just pHRIM620 were selected.

A system of restriction analysis of inserts and sequencing across selected restriction sites was used to obtain a map of pHRIM620.

Inserts that provided complete coverage of pHRIM620 with a good

overlap at each restriction site were selected (FIG. 1). Each of these was tested for their ability to degrade Diuron after growth in M9 media containing Diuron or LB at 25,30 and 37°C for 8,24,48 h (here cells were disrupted by sonication and freeze thaw treatment).

One of the clones, E. coli (pHRIM624) with a large insert (22Kb) was found to degrade Diuron. Best results with this strain were obtained after growth in LB for 24h and three cycles of freeze (-70°C) thaw lysis. After this, Diuron was added and HPLC used to monitor it and any breakdown products over 3 days. These results indicated that the degradative gene (s) on pHRIM624 were expressed from their own promoter (s) in E. coli. The approach used here was dependent on the expression of enzyme activity in E. coli. A few A. globiformis genes have been expressed from their own promoters in E. coli but not all.

Example 4-Identification of the degradative gene (s) and sequence analysis.

Over 150 transposon mutants of pHHRIM624 were tested for their ability to degrade Diuron. A series of mutants were shown to be unable to degrade Diuron and the location of these mutants was determined (FIG. 2). All eight of these insertions were located in two open reading frames within a 2.5 kb SstI fragment.

Other insertions in front of and behind of the genes did not disrupt enzyme activity. Digestion of pHRIM624 with Not I followed by re- ligation, deleted a 12Kb region from one end of the insert and this clone when expressed retained the ability to degrade Diuron.

Digestion of pHRIM624 with SstI and religation provided a clone with a smaller insert with the 2.5Kb SstI fragment, and was able to degrade Diuron. The 2.5 Kb SstI fragment was cut out of this clone and inserted into the SstI site in pUCl9. This clone was able to degrade Diuron.

DNA sequence analysis indicated that there are two open reading frames, one in the positive strand (ORF2-Annex III) and one in the negative strand (ORF4-Annex II).

Since the negative strand contains a predicted ribosome binding site it is most likely that this encodes the phenylurea hydrolase which cleaved Diuron (ORF 4) This reading frame is 1368bp and showed little similarity to any other sequence on the EMBL database. The gene was predicted to code for a 456 amino acid protein with an estimated size of around 49 kDa.

The protein sequence showed a low level of sequence similarity (ca.

25% over 200 aa) to other herbicide hydrolysing enzymes.

Although ORF 2 does not show significant similarity to known DNA and protein sequences, it may still be involved in herbicide breakdown.

The other pesticide degradation genes that have been characterised are involved in the breakdown of 2,4-D, atrazine, carbofuran and parathion (9,17,31,35). These can be complex systems involving upto 8 genes, organised in gene clusters, and located on the chromosome as well as on plasmids (6,13,16). The degradation gene reported in our study bears greater resemblance to simpler systems such as the single genes which encode carbofuran hydrolase (mcd) and parathion hydrolase (pah). In a similar way these single gene systems all have broad substrate specificity. We have named the gene identified in this study puhA, as it encodes for a phenylurea hydrolase and cleaves the carbonyl bond in this group of herbicides.

Methods Bacterial strains and media

A. globiformis D47 and mutants derived from it were routinely grown on Luria Bertrani (LB) agar (Merck, Poole, UK) at 30°C for 2 days and kept at 4°C for up to one month. Fresh cultures from a-70°C stock were regularly obtained to ensure maintenance of the degradative phenotype. For strain selection and degradation assays diuron was added to solutions using the method of Cullington and Walker (7).

Diuron (20mg ml~1 stock solution prepared in 100% methanol) was added to a sterile Schott bottle and the methanol, left to evapourated in a laminar flowbench. Minimal Salts Medium (MSM) or Phosphate Buffered Saline (PBS) was added aseptically and the bottle shaken for 30 min using a wrist action shaker. All E. coli (DH5a, DH10B) clones were routinely grown on LB or in LB broth at 37°C with ampicillin (100 J. g ml-1), trimethoprim (25 Rg ml-1) or kanamycin (50 jj. g ml') to select for cosmid and plasmid vectors, as required.

Plasmid curing of strain D47.

Using 1 ml of an 18 h starter culture of A. globiformis D47 (50 ml LB, 30°C/150rpm), 50 ml of fresh LB was inoculated and incubated at 30°C and 35°C for 24 h. Every 24 h successive transfers were made (1 ml to 50 ml) from each culture for ten days. On transfer, samples were taken (0.1 ml), diluted and plated on to nutrient agar and incubated at 30°C for 2 days. 150 individual colonies were selected and checked for their ability to degrade diuron. A selection (approx.

50) of degradative and non-degradative strains were screened to determine their plasmid composition.

Plasmid DNA profiling and purification.

A small scale plasmid preparation method for profiling strain D47 and its derivatives was used. A single colony was used to inoculate 5ml of LB which was incubated at 30°C for 18h. Cells were collected by centrifugation at 8,000 x g for 10 min and resuspended in 300 pl of buffer PI (Qiagen, Crawley, UK) containing 1mg ml-1 lysozyme. After 10

min at room temperature 300 pl of buffer P2 was added and held on ice for 5 min. To this, 300 fil of P3 was added and the sample centrifuged at 13,000 x g for 15 min to remove the debris. 0.8 vol of isopropanol was added and the sample re-centrifuged at 13,000 x g for 30 mins.

The pellet was washed with 70% (v/v) ethanol and re-suspended in 50 Rl lmM Tris-HC1 (pH8. 0). Samples (usually 20 Rl) were analysed by gel electrophoresis (0.7% w/v agarose). Larger scale purified plasmid preparations were obtained from 500ml cultures in a similar way. The cell pellet was resuspended in 3 ml P1 containing lysosyme and held at room temperature for 15 min and then washed 3 times with PI buffer to remove the lysozyme. After this 3ml volumes of P2 and P3 were added as described above. A QIAGEN tip 100 column was used to clean the DNA, using the manufacturers instructions, and the eluate from the column was isopropanol precipated, washed with 70% (v/v) ethanol and resuspended in a final volume of 100 Rl.

DNA Cloning Plasmid DNA from A. globiformis D47 was digested, individually, with the restriction enzymes EcoRI and SstI. Cut DNA was ligated to the vectors Supercos (Strategene, Amsterdam, The Netherlands) cut with EcoRI, and pUC18 cut with SstI, using standard procedures (Sambrook).

The ligated DNA was heat treated at 65°C for 5 min, dialized and electroporated (12.5 KV cm2) using a BIO-RAD system into E. coli cells. Colonies were selected on LB agar supplemented with kanamycin (Supercos) or ampicillin (pUC18). Over 100 of these colonies were screened using the rapid mini-prep method described above without the addition of lysozyme. The plasmid DNA was digested with either EcoRI or SstI to confirm the presence of inserts and to determine the insert size. Clones with similar sized inserts were grouped, and restriction digestion was used to identify clones that gave an identical pattern. One representative of each plasmid clone with fragment sizes that matched those seen for A. globiformis D47 plasmid DNA was selected. These plasmids were labelled using the Boehringer

digoxigenin kit (Roche, Lewis, UK) and used as hybridization probes.

Target DNA was prepared from degradative, non degradative, and plasmid free strains of A. globiformis D47. Equal quantities (1 Rg) of target DNA was boiled for 5 min, placed on ice for 10 min and spotted onto a nylon membrane (5 Rl). The DNA was fixed to the membrane by UV treatment. For each probe a standard hybridization was carried out. The filter was pre-hybridizated in 6 x SSC containing 0.5% (w/v) Blocking reagent at 72°C for 6 hours. The probe was added and the hybridization continued for 18h. The membrane was successively washed with 2 x, 1 x and 0.2 x SSC containing 0.1% SDS at 72°C for 20 min. The filter was probed and developed using the reagent CPD* using the manufacturers instructions (Boehringer-Roche, UK).

DNA sequencing A system of transposon mutagenesis, sub-cloning from larger fragments, and designing sequencing primers to walk out from known sequences, was used to obtain the sequence of the degradative gene.

All sequencing reactions were performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, UK) and analysed on an automated DNA sequencer (Applied Biosystems). Sequences were edited and assembled using DNA* (DNAStar Inc., Wisconsin, USA) software and sequence analysis was performed using the packages FASTA (6.0). DNA mapping and translation were performed using the programs Clone Manager and Enhance (Scientific & Educational Software, NC, USA).

Transposon mutagenesis Transposon mutagenesis using the artificial transposon AT2 was carried out using a modification of the manufacturers protocol (Applied Biosystems). E. coli plasmid DNA (1, ug in 1 Rl) was mixed with the transposon AT2 in lx buffer (final volume 20 ri) and

incubated at 30°C for 1 hour. The reaction was stopped by the addition of EDTA (final concentration lOmM) and SDS (final concentration 0.05% w/v) and heat treatment (65°C for 15 min). The sample was dialysed by placing 10Ll droplet on a 0.025, um (pore size) filter (Millipore, Watford, UK) floating on H20. After 20 min the sample was electroporated into E. coli cells. Transposon mutants were selected on LB containing 50 Rg ml-1 trimethoprim. Mutants were tested for their ability to degrade diuron. To locate transposon insertion positions in selected mutants, plasmid DNA from mutated clones was purified and the primer island +/-primers (Applied Biosystems) were used to sequence outwards from the transposon ends.

Determination of Diuron degradation by HPLC For A. globiformis D47 and mutants, single colonies were inoculated into sterile glass HPLC vials containing 0.5 ml Mineral Salts Medium and Diuron (20 p. ml-1). The samples were incubated at 30°C for 3 days. To each sample 1 ml of the mobile phase solvent, acetonitrile : water : phosphoric acid (75: 25: 0.25 by volume), was added. The presence of Diuron and any degradation products was determined by HPLC using a Lichrosorb RP18 column (250mm x 4 mm; Merck, UK.) and UV absorbance at 240 nm. For E. coli clones each strain was grown in 5 ml LB containing the appropriate antibiotic at 37°C for 24 h. The cells were collected by centrifugation at 6,000 x g for 5 min and washed twice with phosphate buffered saline (PBS, 0. 1M phosphate pH 7.2,0.85% w/v NaCl). The cell pellet was re-suspended in 100 ttl of PBS and subjected to 3 rounds of freeze thaw treatment (-70°C for 30 min and 30°C for 30 min). To the cell lysate, 10 µg ml-1 diuron was added in PBS and the sample incubated at 30°C for 3 days. Samples were analysed by HPLC to determine the levels of Diuron and DCA. Controls included E. coli, E. coli (pUCl8) and A. globiformis D47.

Table 1 Media Diuron D47 D47 Non- Conc. (µg Degrader degrader ml-1) LB Agar 0 ++++ ++++ .. 10 ++++ ++++ .. 20 +++ +++ .. 30-- MSM 0 + slight + slight .. 10 +slight + slight .. 20 + slight + slight .. 30 - - Table 2 Overlap Acc. No. Strain Enzyme identi (aas) (Reference) ty 23.4 435 Q9S1C6 (22) Arthrobacter Organophosphorus hydrolase sp.

26.0 460 Q50432 (19) Mycobacterium Organophosphate acid sp. anhydrase 25. 7 458 CAC04032 Streptomyces Organophosphate acid (25) sp. anhydrase 24.0 425 BAB06654 Bacillus sp. Aryldialkylphosphatase (32) 24.6 425 027577 (30) Methanobacteri Aryldialkylphosphatase um

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Identification and characterisation of a diuron-degrading bacterium.

ANNEX I 04 Oct 2000 Sequence Data Page 1 Molecule: SstI fragment, 2519 bps DNA Description: product of cut plasmid SstI File Name: (not saved), dated Printed: 1-2519 bps (Full), format Single Strand 1 ccgcacgacc ggcaccgggg cccaggtcat acgcggtcag caccagattg 51 gcctgcgccg gcgtggcgat acccgcctgg tggatgccgt cgaacggcac 101 ggagtcgcgc gcgtcggcga ccggcgacgg ggctccgtcg gcggccggtg 151 ccgccggcgc ggtcagacgt ccgactgtcg cgcccgacgc gaatccggtg 201 attccgacga ccgccccggc accgacgagg ccgaacaggc tccgacgact 251 gacggcagag ccaccgccct tcgcggcgga aggctcggtc gcggtgtcgc 301 cgcccgatcg ggtcacggcc ggatccgctc gcccgacggc gtcagcgccc 351 accaggtgcc accgttctgg tcgcgaccgg acccggtcac ctgccccagc 401 tggtcggagg tgtagcggta gagcggccag ccgtcgaagg tggccacgtc 451 gacgccgtcc tggtcggcga tgacgccgac acggcctgcc ccgatacccg 501 cggcgaccat gggctcggcg cccgcgtcca ccatcagcgg cggccacttg 551 gccgcgcatc cgccggtgca ggtgacgtcc ttggcgtcgt cgggttcgaa 601 gatgtacagc gcctggccgt cgccgtcgac gaggacctcg cccatgccgt 651 cgacggatgc gacctcgacg gctgcgggac tgtcgtcggt cacgggccgt 701 tcggccgtca gaacgaagaa cgcggtcgcg gatccgacga cgagcgcggc 751 accgacggtg ccggcgacga cacgagtgat gatgcgtcgc cgccggctgg 801 cctcggggcg atccgaagga ggggtatcga gctggggact ggtgagttcg 851 gtcatgtgtt agatgctctc acagagttac agcttctaac aagacccatc 901 tcgccgctcg gcggcgagat ggcgttgtca ggcgggatgc gccgtgacga 951 gcggcgtggt gggcagggca ccacggtcga cggcgacacc ggcctggtac 1001 acggcgctga tggagcgcag gtgacggatg tcggcggtgg ggtcggcgtc 1051 gagcagcacg aagtccgcga gcttgcccgt ctcgaccgag ccgatgtcgg 1101 catccttgcc gtaggcgcgc gcgacgttca gcgtcgccgc cgagatcgcc 1151 tccagcSgggg tcatgccctt ctccacgatg gactgggtcc agtggaagtg 1201 gtcgccgccg atcgtccagg gtcgatcctc gcgctccgcg ggcgacaggt 1251 cggcgaggtg atccttgctc gggcagccgg catcggtgtt catgaggatc 1301 ttcgccttgg cacggatcaa cgcgcgctcg ttcgacgcgt acggctcccc 1351 cgcgagcgtc gccgcccagc ttccgacccg ctcgagcccc tcacggtgct 1401 cgtggtgcac ggtctgcagc cccgcgtaag agtcgctcgc ggcgatcttg 1451 tcgatgaggt agtccgggat gtgctgtccg agcgtgtagt tcgcgtggat 1501 caggacgtcg gcaccgaggt cgaccgaggt gtcgagcgcc tccaccgaca 1551 cgctgtgcgt cagcaccggc acccccgcag ctcgcgcttc ctcgaacatg 1601 acctcgagca cggggcggct gaaggtctgg cacgagcggt cgaacccgac 1651 ggtgaagacg atgtggtcgc tgacggcgat cttcagcatg tcgacgccgc 1701 gagcgagata gtcccgcacg cgcgaacgca cctcgttgcg gggcaggagg 1751 ctcagttgat gccccacccc ggcctcgaac atggcgtcga tgcggttgac 1801 gaacgtccgc gacgccgcct gctgcccgat gaagtggaaa tcggcgctga 1851 acggtccgcc caggccgacg atggttcccg cggcgaagat gcgggcgccg 1901 gcggaggtgc ccgcgttgat gcggtcgcgc gcggccagca cgggctcgat 1951 cgcgttgtac gtgtcgaaca cggtcgtcac gccgttgcgc agcacgagct 2001 gcgcggcttc ctcgatcacc tcgacgtagc gcccctccca ccgcgcgagg 2051 tactcgatcg tgccgggccc gaccatgaac atccatgcgt cgaggaggtg 2101 cacattgccg ttgacgtagc ccggcaccat ccaccgaccg gcgccgccga 2151 cgacgcgggc gccgtcgggg atgggcgtgg tggcggtcgg gccgatggcc 2201 gagaagcggt cctcgtcgac gagaacggtc actccgcgtt cgggcgcccc 2251 tcctcgtcca tcgatcaggg tgatgtcggt gatggcggtg gtggtcatgg 2301 aacacgtcct cgagttcggt ggcgccggtc aggtgccctc agactaaccg 2351 aaccgatcgg ttctgtaaac cactcggttc ggatacatcg gtagagtgcc 2401 ttcatgccac acgagcagat cgaggtcgtc gacgggcgca. cacgccggtg 2451 ggagggccgc aagagcgacc tcgtccacgc ggcggtcgag tacgtcctgg 2501 acaccggcat cgccgagct ANNEX II 04 Oct 2000 Sequence Data Page 1 Molecule: protein orf4,456 aas Protein Description: Translation of SstI Fragment starting at bp 2298 (Complem strand) File Name: (not saved), dated Printed: 1-456 aas (Full), format Single Strand 1 mtttaitdit lidgrggape rgvtvlvded rfsaigptat tpipdgarvv 51 ggagrwmvpg yvngnvhlld awmfmvgpgt ieylarwegr yvevieeaaq 101 lvlrngvttv fdtynaiepv laardrinag tsagarifaa gtivglggpf 151 sadfhfigqq aasrtfvnri damfeagvgh qlsllprnev rsrvrdylar 201 gvdmlkiavs dhivftvgfd rscqtfsrpv levmfeeara agvpvlthsv 251 svealdtsvd lgadvlihan ytlgqhipdy lidkiaasds yaglqtvhhe 301 hreglervgs waatlagepy asneralira kakilmntda gcpskdhlad 351 lspaeredrp wtiggdhfhw tqsivekgmt pleaisaatl nvaraygkda 401 digsvetgkl adfvlldadp tadirhlrsi savyqagvav drgalpttpl 451 vtahpa ANNEX III 04 Oct 2000 Sequence Data Page 1 Molecule: Protein orf2,491 aas Protein Description: Translation of SstI Fragment starting at bp 2458 (Complem strand) File Name: degpro2. cm5, dated 19 Sep 2000 Printed: 1-491 aas (Full), format Single Strand 1 malsggmrrd errggqgttv dgdtglvhga dgaqvtdvgg gvgveqhevr 51 elarldradv gilavgardv qrrrrdrlqr ghallhdglg pvevvaadrp 101 gsilalrgrq vgevilaraa gigvhedlrl gtdqralvrr vrlprerrrp 151 asdplepltv lvvhglqprv rvargdlvde vvrdvlserv vrvdqdvgte 201 vdrgverlhr havrqhrhpr ssrflehdle hgaaeglara vepdgeddvv 251 adgdlqhvda aseivphart hlvagqeaql mphpglehgv davderprrr 301 llpdeveiga ersaqaddgs rgedagaggg arvdavargq hgldrvvrve 351 hgrhavaqhe lrgfldhldv aplpprevld ragpdhehpc veevhiavdv 401 arhhpptgaa ddagavgdgr gggradgrea vlvdenghsa fgrpsssidq 451 gdvgdggggh gtrprvrwrr sgalrltepi gsvnhsvrih r