NZ274482A - Cells transformed with a truncated and/or mutated staphylococcus aureus nuclease and their use in immunological, pesticidal and environmental pollutant-degrading compositions - Google Patents

Description

New Zealand No. 274482 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 13.10.1993; Complete Specification Filed: 12.10.1994 Classification:^) C12N5/10; C12N15/55.70; C12N1/21; C12N9/22; A01N63/00; C12S1/00 Publication date: 22 September 1997 Journal No.: 1420 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Recombinant staphylococcal nucleases and their use in limiting the survival of genetically engineered microorganisms Name, address and nationality of applicant(s) as in international application form: GX BIOSYSTEMS A/S, Forskerbyen Symbion, Fruebjergvej 3, DK-2100 Copenhagen O, Denmark New Zealand No. 274482 International No. PCT/DK94/00381 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Recombinant staphylococcal nucleases and their use in limiting the survival of genetically engineered microorganisms Name, address and nationality of applicant(s) as in international application form: GX BIOSYSTEMS A/S, of Forskerbyen Symbion, Fruebjergvej 3, DK-2100 Copenhagen 0, Denmark f ci be/VWCVl* ■ company £) WO 95/10614 1 274482 RECOMBINANT STAPHYLOCOCCAL NUCLEASES AND THEIR USE IN LIMITING THE SURVIVAL OF GENETICALLY ENGINEERED MICROORGANISMS FIELD OF INVENTION The present invention provides, a novel system of biological 5 containment of genetically engineered microorganisms. In particular, the invention relates to novel recombinant staphylococcal nucleases; to a cell or a population hereof containing a regulatably expressible gene whose expression results in the formation of the cytoplasmatically active 10 staphylococcal nuclease at a rate which leads to cell function limitation; to a recombinant replicon containing such a regulatably expressible gene; to methods of limiting the survival of a population of cells containing the expressible gene, to a method of containing an extrachromosomal recombi-15 nant replicon to a first kind of cells and a method of stochastically limiting the survival of a cell population.

There is also provided an immunologically active composition, a pesticidal composition and an environmental pollutant-degrading composition, all of which contain a cell population 20 as defined above wherein the cells further contain a DNA sequence coding for a gene product which is immunologically active, pesticidally active or environmental pollutant-degrading, respectively.

TECHNICAL BACKGROUND The increasing application of recombinant DNA technology to engineer novel microorganism which are industrially useful have caused concerns in both the scientific community and the general public over potential risks. These concerns are primarily related to the potential harm to humans and to 30 undesirable and/or uncontrollable ecological consequences upon deliberate or unintentional release of such genetically engineered microorganisms (GEMs) into the environment. These ^WO 95/10614 PCT/DK94/00381 2 concerns have led to the establishment of official guidelines for the safe handling of GEMs in laboratories and production facilities where such organisms are applied. Up till now, such guidelines have primarily been directed to measures of 5 physically containing GEMs in laboratories and production facilities with the aim of reducing the likelihood that workers in such facilities were contaminated, or that the GEMs were to escape from their primary physical environment, such as a fermentation vessel.

It is presently being recognized that the level of safety in the handling of GEMs can be increased by combining physical containment measures with biological containment measures to reduce the possibility of the survival of the genetically engineered organisms if they were to escape from their pri-15 mary environment.

Lately, however, concerns have become increasingly focused on potential risks related to deliberate release of GEMs to the outer environment and to the use of GEMs as live vaccines. In this connection there is a strongly felt need to have bio-20 logical containment systems which subsequent to the environmental release of the GEMs or their administration as vaccines to a human or an animal body, effectively kill the released organisms in a controlled way or which limit the function of the released GEMs to an extent where such GEMs 25 are placed at a significant competitive disadvantage whereby they will eventually be ousted by the natural microflora of the environment to which they are released.

The first systems of biological containment were based on the use of "safe" cloning vectors and debilitated host bacteria. 30 As examples, it has been suggested to select vectors which lack transfer functions or which naturally have a very narrow host range. Examples of debilitated host bacteria are E. coli mutants having an obligate requirement for exogenous nutrients not present or present in low concentrations outside the 35 primary environment of the GEMs. 3 Other suggested biological containment systems have been based on mechanisms whereby the vector is restricted to the GEMs e.g. by using a plasmid vector with a non-sense mutation in a gene, the expression of which is indispensable for 5 plasmid replication or a suppressor mutation in the chromosome, said mutation blocking txanslational read-through of the message of the gene. A further approach is to maintain the rDNA stably in the host by integrating it into the chromosomes of the GEMs.

Recently, an alternative biological containment strategy has been developed in which the recombinant vector is endowed with a gene encoding a cell killing function which gene is under the control of a promoter only being expressed under certain environmental conditions, such as conditions prevail-15 ing in an environment outside the primary environment of the GEMs, or when the vector is unintentionally transferred to a secondary host, or the expression of which is stochastically induced. By using incorporation in a GEM of such a cell killing function and selecting appropriate regulatory 20 sequences, vectors can be constructed which are contained in the primary host cell and/or in a primary physical environment. A cell killing function as hereindefined may also be referred to as an active biological containment factor.

If a stochastically induced mechanism of expression regula-25 tion is selected for such a biological containment system, a population of GEMs containing the system will, upon release to the outer environment, or if used as a live vaccine be subjected to a random cell killing which will lead to an increase of the doubling time of the host cell population or 30 eventually to the disappearance of the organisms.

The above-mentioned genes encoding cell killing functions are also frequently referred to as "suicide" genes, and biological containment systems based upon the use of such genes, the expression of which are regulated as defined above, are 35 commonly described as conditional lethal systems or "suicide" WO 95/10614 PCT/DK94/00381 4 systems. Up till now, several cell killing functions have been found in bacterial chromosomes and in prokaryotic plas-mids. Examples of chromosomal genes having cell killing functions are the gef (Poulsen et a.l,, 1990) and re IF (Bech 5 et al., 1985, Embo, vol.4 no.4, 1059-1066) genes from E.coli K-12. Examples of plasmid encoded suicide genes are hok and flmA. (Gerdes et al., 1986) genes isolated from plasmids R1 and F, respectively, the sjotB gene also isolated from plasmid F (Akimoto et al., 1986) and the pnd gene isolated from 10 plasmids R16 and R483 (Sakikawa et al., 1989 and Ono et al., 1987). Common features of these genes are that they are transcribed constitutively, regulated at a post-transcriptional level, and that they all encode small toxic proteins of about 50 amino acids. The application of the hok gene in a 15 biological containment system has been disclosed in WO 87/05932.

Ideally, the features of an effective biological containment system should include as a minimum requirement that the cell killing function when expressed is effective, that the con-20 tainment system is functional in a broad range of species of GEMs, that the risk of elimination of the cell killing function e.g. by mutations in the suicide gene or the sequences regulating the expression of the gene, is minimal and that the risk of uptake by other organisms of rDNA. released when 25 cells are killed is reduced.

None of the above-mentioned known containment systems fulfil all of these ideal requirements. However, in applicants' copending application WO 93/20211 there is provided an active biological containment system which is not based on a primary 30 cell killing function but which makes use of genes, the expression of which in a cell where the gene is inserted, results in the formation of mature forms of exoenzymes such as nucleases derived from gram-negative or gram-positive bacteria or phospholipases which are hydrolytically active in 35 the cytoplasm of the cell and which can not be transported over the cell membrane. When such enzymes are expressed in a cell, the normal function of the cell becomes limited to an extent whereby the competitiveness, and hence the survival and the environmental persistence, of a population of such cells are reduced significantly.

However, it has been observed .that although a bacterial nuclease encoded by a gene from which the sequence coding for the signal peptide has been totally removed may be useful in a method of limiting the survival of GEMs in accordance with the above, a certain transport of the nuclease out of the 10 cell comprising such a gene without the signal sequence- coding sequence may caube "leakage" of active enzyme out of the cell, thereby reducing the efficiency of this containment system. It has now surprisingly been found that this problem can be overcome if a truncated and/or mutated form of a 15 nuclease is used. Additionally, a significant increase in the cytoplasmic enzymatic activity of such a modified enzyme relative to the parent enzyme has been provided thus resulting in improved methods of limiting the survival of GEMs.

Provided the hydrolytically active enzyme is a RNA-degrading 20 and/or DNA-degrading enzyme, a biological containment system based upon such an enzyme may have a further advantage over the known biological containment systems in that the rDNA molecules in a genetically engineered host cell is destroyed * simultaneously with the genetically altered host microorga-25 nisms. As an example, a recombinant staphylococcal nuclease according the invention having endonucleolytic activity will when used in accordance with the invention cause hydrolysis of the DNA and the RNA to 3'-phosphomononucleotides. However, it may from a GEM risk point of view be advantageous to 30 provide the GEM containment system in a form where further intracellular degradation of the damaged nucleic acids is provided. This may e.g. be provided by inserting into the GEMs a further gene coding for a 3'-5' exonucleolytically active exonuclease. The simultaneous expression and the 35 successive degradative activities of a endonuclease such as the above modified staphylococcal nuclease and an exonuclease ^ WO 95/10614 6 will cause complete destruction of the host nucleic acids, thus killing the host strain without having its nucleic acids remaining stable in the environment for an unlimited period of time.

SUMMARY OF THE INVENTION Many cells produce hydrolytically active enzymes which are inherently translocated extracellularly, i.e. the enzymes are excreted over the cell membrane. When expressed intracellu-10 larly in a cell naturally producing such enzymes, the enzymes are in the form of enzymatically inactive, immature exoenzyme molecules (proenzymes) comprising a signal peptide by means of which the proenzyme is transportable over the cell membrane. On passage of the cell membrane, the signal peptide is 15 cleaved off the molecules which are thereby turned into the mature, enzymatically active form.

The present invention is based on the discovery that a modified, i.e. a truncated and/or mutated form of Staphylococcus aureus nuclease being an exoenzyme which normally, while 20 present in the cytoplasm of cells producing them, are in the form of the immature, enzymatically inactive proenzyme may be expressed in a cell in the form of a truncated and/or mutated form of the mature enzyme which is enzymatically active in the cytoplasm of the cell and that the presence intracellu-25 larly of such active forms of such modified staphylococcal nucleases may cause damages to the cells leading to a limitation of the normal cell function. 7 27 4 4B 2 On the basis of these findings, the present invention provides a novel improved approach to biological containment of GEMs aijming at limiting the survival hereof in their environment.

Thus, in a first aspect, the present invention provides a cell containing a regulatably expressible gene coding for a truncated and/or mutated Staphylococcus aureus nuclease capable of Intracellularly hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell membrane, the cell further containing a regulatory nucleotide sequence which regulates the expression of the gene.

In a further aspect, there is provided a recombinant replicon comprising a regulatably expressible gene coding for a truncated and/or mutated Staphylococcus aureus nuclease capable of intracellularly hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in a cell comprising the replicon, essentially cannot be translocated outside the cell membrane, the expression of said gene being regulatable by a regulatory nucleotide sequence which is contained in the recombinant replicon or in another recombinant replicon present in a cell containing the replicon.

In a still further aspect, the present invention relates to a population of cells consisting of a multiplicity of cells as defined above.

Additionally, the present invention relates to a method of limiting the survival of a cell population in a first or a second environment which method comprises transforming the 8 cells of said population with a recombinant replicon being replicated in the cells of the population and containing a gene whose expression results in the formation of a truncated and/or mutated Staphylococcus aureus nuclease which is pres-5 ent and hydrolytically active in the cytoplasm of said cell, the cells further containing a. regulatory nucleotide sequence being regulatable by an environmental factor and which regulates the expression of said gene, the expression of said gene leading to formation of the enzyme in the cells at a 10 rate which results in the hydrolysis of hydrolysable cytoplasmic substances necessary for non-limited function of the cell, to an extent whereby the function of the cells is being limited leading to a limitation of the survival of the cell population.

In another aspect, the invention relates to a method of containing an extrachromosomal recombinant replicon to a first kind of cell, where said replicon is naturally transferable to a second kind of cell, which method comprises providing on the recombinant extrachromosomal replicon a gene 20 whose expression results in the formation of a truncated and/or mutated Staphylococcus aureus nuclease which is present and hydrolytically active in the cytoplasm of a cell, the formation of said enzyme being at a rate which results in the hydrolysis of hydrolysable cytoplasmic substances necessary 25 for non-limited function of the cell, to an extent whereby the function of the cell is being limited, said first kind of cells having or being modified to have a chromosomal replicon comprising a regulatory nucleotide sequence which inhibits the expression of said gene and thereby protects said first 30 kind of cells, said regulatory gene being lacking in said second kind of cell, whereby, if a cell of the second kind receives said extrachromosomal recombinant replicon said gene is expressed and has a function-limiting effect thereon.

In another further aspect, the present invention pertains to 35 a method of stochastically limiting the survival of a cell population which comprises transforming the cells thereof 9 with a recombinant replicon containing a regulatably expressible gene which, when expressed in a cell encodes a truncated and/or mutated Staphylococcus aureus nuclease which is present and hydrolytically active in the cytoplasm of the 5 cell, the expression of said gene leading to formation of the enzywe in the cell at a rate which results in the hydrolysis of hydrolysable cytoplasmic substances necessary for non-limited function of the cells, to an extent whereby the function of the cells is being limited, the expression of 0 said genes or genes being stochastically induced as a result of recombinational excision of an excisable negatively functioning regulatory nucleotide sequence which, while present in the cells, inhibits expression of the gene coding for the enzyme, said negatively functioning regulatory nucleotide 5 sequence being contained in the recombinant replicon or in an other recombinant replicon present in cells of the population containing the replicon.

In a still further aspect, there is provided an immunologically active composition which contains a viable function-0 limited cell population as defined above wherein the cells contain a further DNA sequence not naturally related to the gene coding for the hydrolytically active enzyme or to the regulatory nucleotide sequence, which further sequence is a sequence coding for an immunologically active gene product, 5 the cells being function-limited to an extent which, wheii the composition is administered to a human or an animal, allows the cells to express the immunologically active gene product for a period of time and in an amount sufficient to obtain an effective immune response in said human or animal, but which 0 does not ".How the cells to persist in the human or the animal.

The present invention also provides a pesticidally active composition which contains a viable cell population as defined herein wherein the cells contain a further DNA 5 sequence not naturally related to the gene coding for the hydrolytically active enzyme or to the regulatory nucleotide sequence, which further sequence is a sequence coding for a pesticidally active gene product, the cells being function-limited to an extent which, when cLe composition is administered to an environment containing a pest, allows the cells 5 to express the pesticidally active gene product for a period of time and in an amount sufficient to obtain an effective pesticidal effect in said environment but which does not allow the cells to persist in the environment.

Finally, there is provided an environmental pollutant - degra-10 ding composition which contains a viable cell population as defined herein, wherein the cells contain a further DNA sequence not naturally related to the gene coding for the hydrolytically active enzyme or to the regulatory nucleotide sequence, which further sequence is a sequence coding for an 15 environmental pollutant-degrading gene product, the cells being function-limited to an extent which, when the composition is administered to an environment containing a pollutant to be degraded, allows the cells to express said pollutant degrading gene product for a period of time and in an amount 20 sufficient to obtain an effective pollutant-degrading effect in said environment but which does not allow the cells to persist in the environment.

DETAILED DISCLOSURE OF THE INVENTION The concept of an active biological containment system based 25 on the use of an intracellularly hvdrolvticallv active exoenzyme which has a cell function-limiting effect A number of macromolecules such as proteins, lipids, phospholipids, DNA, RNA and polysaccharides that cannot pass cell membranes are nevertheless utilizable as substrates for cell 30 growth. These substrates are enzymatically hydrolysed (degraded) in the external medium of the cells by protein enzymes excreted by the cells. Such hydrolytic enzymes that mediate extracellular degradation are termed "exoenzymes" . In addi 11 tion to true exoenzymes which are capable of passing the outer cell membrcine and hence excreted into the external xn^dium, certain cells produce incompletely excreted hydrolytic enzymes of which some enter the cell membrane and remain 5 fch^re rather than passing through. Others pass through the cell membrane but not the outer membrane (periplasmic enzymes) and still others remain in the outer membrane.

In the present context, the term "exoenzyme" is used to designate a hydrolytic enzyme which have been excreted natu-10 rally by the cell natively producing the enzyme or a cell in which the genes coding for such an excretable enzyme has been inserted, by passing through the cell membrane (the cytoplasmic membrane) and accordingly, this term include native true exoenzymes, cell membrane-bound enzymes, periplasmically 15 located enzymes and outer membrane-bound enzymes.

Generally, the mechanisms by which excretable exoenzymes as defined herein are excreted share common features. Thus, when first synthesized in the cytoplasm, these proteins are in the form of enzymatically inactive precursor molecules (also 20 referred to as proenzymes) which are larger than they are after they have entered their proper extracellular location, since a portion of the amino-terminal end of the protein which is termed as the signal peptide, is removed after the protein is properly located. The function of the signal 25 peptide is to aid in the translocation of the protein exoenzyme to its place of location. When the molecule is properly located, the signal peptide is removed whereby the protein is turned into the mature or "processed", enzymatically active hydrolytic enzyme.

As explained above, the present invention presents a novel approach to the concept of active biological containment which is based on the finding that it is possible to obtain in the cytoplasm of a cell, i.e. intracellularly, the presence of enzymatically active mature forms of exoenzymes. This 35 is obtained by the insertion in the cell of a nucleotide WO 95/10614 PCT/DK94/00381 12 sequence which codes for the exoenzyme molecule, but which does not comprise the sequence coding for the corresponding native signal peptide herefor. By deletion from the nucleotide sequence of the sequence coding for the signal peptide 5 it is also obtained that the intracellularly active enzyme molecule cannot be translocated outside the cell membrane.

The cytoplasm of cells contains a mixture of macromolecules, the presence and function of which are necessary for the non-limited function of the cells, including basic life mani-10 festations such as growth and replication. Examples of such macromolecules include DNA, RNA, lipids, phospholipids, proteins and polymeric carbohydrates.

When, in accordance with the present invention, a gene whose expression results in the formation of an exoenzyme as 15 defined above is inserted into the cell, naturally occurring intracellular macromolecules may act as substrate for the intracellularly active hydrolytic enzyme.

Provided the hydrolytically active enzyme is expressed intracellularly at a rate which results in the hydrolysis of 20 hydrolysable cytoplasmic macromolecules to an extent whereby the life functions of the cells become limited, the competitiveness of such cells with cells of the same type, but in which an exoenzyme as defined herein is not expressed in intracellularly active forms, will be decreased.

In the present context, the term "non-limited cell function5 denotes that the growth of a cell as manifested i.a. by the synthesis of new cell material and the rate of replication of the cell is not decreased by an intracellular hydrolytic macromolecule degradation, not natively occurring in the 30 cells. Accordingly, this term is used herein to describe i.a. the growth rate and the rate of replication, under any given condition, of a cell in which an intracellularly active exoenzyme as defined above is not being expressed. Consequently, the term "limited cell function" as used herein WO 95/10614 PCT/DK94/00381 13 describes, in a relative manner, the state of a cell in terms of i.a. a reduced growth rate and/or reduced rate of replication which results from the expression therein of an intracellularly active exoenzyme when the cell is grown under the 5 same conditions as a cell of the same kind having a non-limited cell function as defined above.

The recognizable manifestation of such limited cell function may ultimately be cell death, but it may also, relative to a cell having a non-limited cell function, be a reduced cell 10 growth appearing as a reduced rate of replication resulting in a reduced increase of cell numbers within a certain period of time as a result of an increase of the lag phase and/or of the cell doubling time. Other manifestations may be a relatively increased requirement for one or more nutrient conqpo-15 nents or a relatively higher susceptibility to detrimental environmental factors such as sub-optimal temperatures or cell damaging caused by toxic substances.

The actual type of manifestation or manifestations of such limited cell function and the degree hereof will depend in 20 particular on the specific species of the exoenzyme being expressed in the cell, the rate of expression of the exoenzyme, the capability of the enzyme to be hydrolytically active under the conditions prevailing in the cytoplasm, the amounts of substrate macromolecules, and the significance of 25 such macromolecules for non-limited function of the cell, and the number of copies of the gene encoding the intracellularly active exoenzyme.

The intracellularly hvdrolvticallv active exoenzvme having cell function-limiting effect In accordance with the invention, a suitable intracellularly active exoenzyme may be any truncated and/or mutated Staphylococcus aureus nuclease which, when it is expressed, is capable of hydrolytically degrading macromolecules present in 14 the cytoplasm of prokaryotic and eucaryotic cells to an extent whereby the function of the cell is being limited.

As used herein, the term "nuclease" denotes hydrolytic exoenzymes capable of degrading nucleic acids. The nucleic acids 5 DNA and RNA are polynucleotides formed by the joining of nucleotides by phosphodiester bonds. Some nucleases are capable of degrading both DNA and RNA, whereas others (deoxy-ribonucleases or DNases) degrade only DNA and still others degrade only RNA (ribon"~leases or RNases). Nucleases may 0 either be exonucleases which are diesterphosphate bond- hydrolysing enzymes cleaving off the last nucleotide residue in either of the two terminals of em oligonucleotide, or endonucleases which cleave phosphodiester bonds located in the interior of polynucleotides. In the art, the term "nick" 5 is normally used to describe the lesion in a DNA strand resulting from an endonuclease cleavage of a diesterphosphate bond. It must be noted that term "exonuclease" when used in the present specific context denotes the mode of action as defined above and thus, the term is not used here to desig-0 nate a nuclease normally being excreted out of the cell.

In the context of the present invention, useful nucleases include staphylococcal nucleases having the capability of hydrolysing diesterphosphate bonds in DNA. Useful DNA-degrading nucleases include endonucleases, the activity of which, 5 when double-stranded DNA is the substrate, results in the cleavage of diesterphosphate bonds (nicks) in only one of the strands of the DNA. Accordingly, in one embodiment of the invention, the hydrolytically active enzyme is a staphylococcal endonuclease having such capability. 0 Lesions in the DNA occur spontaneously in all cells with a relatively high frequency. However, in the normal cell such lesions are efficiently eliminated by an inherent DNA repair mechanism which involves that the altered portion of the damaged strand is recognized and removed by one set of en-5 zymes and then replaced in its original form by a DNA poly- WO 95/10614 PCT/DK94/00381 me.rase, and finally an enzyme called DNA ligase seals the nick (the broken diesterphosphate bond) that remains in the DNA helix, to complete the restoration of an intact DNA strand.

This natural cell DNA repair mechanism will also eliminate DNA lesions (nicks) which may result from the enzymatic activity in a cell of a nuclease as defined herein. Accordingly, the function of a cell in which the intracellularly active nuclease as defined above, is expressed will only be 10 limited when the nuclease is expressed at a rate which results in the presence of nicks in the cell nucleic acids in a number exceeding the number which can be repaired by the DNA repair mechanism, so that the number of DNA lesions (nicks) reaches a level whereby the function of the cell becomes 15 recognizably limited.

The rate of expression of the intracellularly active exoenzyme as defined herein is also determined by the specific activity of the enzyme under the conditions prevailing in the cytoplasm of the cell. It will be understood that these 20 conditions may differ from those prevailing in the extracellular environment into which the enzyme when present in a cell where it is normally produced, is translocated. As one example, an enzyme comprising sulphur-containing amino acid residues such as cysteine and methionine will be prone to 25 damages caused by oxidation and hence, it may be assumed that it will be at least partially enzymatically inactive when present in the cytoplasm of a cell where the Eh-value is high. However, it was surprisingly found that an enzyme comprising sulphur-containing amino acids may be enzymatical-30 ly active intracellularly to an extent whereby the function of a cell in which the enzyme is expressed, is limited.

Several prokaryotic cells including gram-negative and gram-positive bacteria inherently produce exoenzymes which are translocated over the cell membrane, interesting examples 35 being endonucleases produced by Serratia spp. It is known WO 95/10614 PCT/DK94/00381 16 that such endonucleases have a high specific activity ex-tracellularly. In WO 86/06743 are disclosed such extracellular Serratia spp. nucleases being expressed in E. coli and their usefulness in the removal of nucleic acids from bi-5 ological materials is described. In WO 93/20211 is disclosed a Serratia spp. nuclease which when expressed intracellularly in the form of the mature enzyme, i.e. without the signal peptide, is enzymatically active to an extent whereby the function of the cell in which the enzyme is expressed, may be 10 limited.

The present invention pertains to a hydrolytically active enzyme which is a truncated and/or mutated form of a nuclease encoded by a gene isolated from a gram-positive bacterial species such as a Staphylococcus species, one suitable 15 example hereof being the mature form of the nuclease of the Foggi strain of Staphylococcus aureus (ATTC 27355). The gene encoding this enzyme has been cloned and characterized (Shortle, 1983, Gene, 22, 181-189) . This nuclease is a Ca2+-activated 16.8 kD thermostable extracellular phosphodiester-20 ase which degrades both DNA and RNA. Itr enzyme consists of 149 amino acids with no disulphide bonds or free sulfhydryl groups. The gene encoding this wildtype nuclease may be derived from plasmid pFOG408 or plasmid pFOGSOl containing the gene.

During the experimentation leading to the present invention, it has been found that a staphylococcal nuclease including the above nuclease of the Staphylococcus aureus (Foggi) strain may be hydrolytically active even in a truncated form, i.e. in a form where amino acid residues have been deleted 30 from the N-terminal or the C-terminal end of the wild type enzyme, or from both ends. Examples of such truncated hydrolytically active enzymes are nucleases encoded by the plasmids pSNUC24-26 and pSNUC420-26 as described hereinbelow. Such truncated enzymes may have improved characteristics as 35 compared to the parent enzymes from which they are derived. Such improved characteristics may include higher specific 17 activity, higher intracellular stability, no or very low transport of the enzyme out of the cell.

Accordingly, in other useful embodiments of the present invention the hydrolytically active enzyme is in a truncated 5 form of a staphylococcal endonuclease, e.g. lacking at least 9 amino acid residues of the parent enzyme from which it is derived.

Such a truncated staphylococcal nuclease may even maintain its enzymatical functionality when further modified relative 10 to the parent enzyme. Thus, such a truncated nuclease may be mutated in one or more codons by substitution of at least one nucleotide or it may be modified by insertion of DNA sequences in the coding sequence for the truncated enzyme. Furthermore, it was found that deletions of one or more 15 nucleotides may occur in the coding sequence without impairing the function of the enzyme, even when such a deletion causes a frameshift mutation.

A cell containing the hvdrolvticallv active exoenzvme As mentioned above, the present invention relates in one 20 aspect to a cell containing a gene coding for the hydrolytically active exoenzyme as defined herein. The cell may be selected from a wide variety of cells for which a need for containment exists. Thus, the cell to be contained may be a bacterial cell, a protozoan cell, a yeast or fungal cell, or 25 a cell derived from the tissues of multicellular organisms such as plants, animal and fungi.

The gene coding for the hydrolytically active enzyme may be derived from a variety of replicons contained in any Staphylococcus organism producing an extracellular enzyme as pre-30 sently defined. Thus, sources of the gene include staphylococcal chromosomes or plasmids. The gene may also be constructed synthetically according to standard procedures. 18 In accordance with the invention, the cell as defined herein may be obtained by methods known per se. These methods include the steps of screening for cells expressing a suitable extracellular hydrolytic enzyme, isolating from such cells 5 the gene encoding the enzyme, removing from the gene the nucleotide sequence coding for the signal peptide and, if desired, removing part of the coding sequence for the mature form of the nuclease, and/or mutating the gene in one or more codons, and inserting into a cell to be contained, the thus 10 modified gene and a nucleotide sequence capable of regulating the expression hereof, growing the cell under conditions allowing the expression of the gene, and testing for the intracellular presence of the enzyme in an enzymatically active form.

The insertion of the gene coding for the hydrolytically active exoenzyme may be in the chromosome of the cell or the gene may be inserted in an extrachromosomal recombinant replicon such as a recombinant plasmid capable of replicating in the cell.

The regulatory nucleotide sequence regulating the expression of the .wdrolvticallv active exoenzvme As mentioned above, the expression of the gene coding for the hydrolytically active enzyme is regulated by a regulatory nucleotide sequence. In the present context the term "regula-25 tory nucleotide sequence" is intended to indicate a nucleotide sequence which directly or indirectly regulates the expression of the gene coding for the hydrolytically active enzyme at the level of transcription or at the level of translation. The regulatory nucleotide sequence may be one, 30 the function of which results in a suppression or inhibition of the activity of the regulatable promoter. Such regulatory nucleotide sequences are herein referred to as "negatively functioning regulatory nucleotide sequences". 19 One interesting example of such a negatively functioning regulatory nucleotide sequence is a sequence coding for a repressor substance which represses the expression of the gene coding for the hydrolytically active enzyme and which 5 substance may, when a cell containing it is released to a human or an animal body or to .the outer environment, undergo a decay whereby the repression of expression of the enzyme-encoding gene is gradually reduced and eventually, when the decay of the repressor is completed, the repression is remo-10 ved.

In preferred embodiments of the invention, the regulatory nucleotide sequence may be contained in the cell in one or more recombinant replicons and it may be contained in the same replicon as that containing the enzyme-encoding gene or 15 in a different recombinant replicon.

One way whereby the expression of the cell function-limiting enzyme may in accordance with the invention be regulated is by providing in the cell a gene coding for the hydrolytically active enzyme, which gene is regulated at the level of trans-20 cription. The regulation at the level of transcription may be carried out in various ways including a regulation by means of a promoter, regulated by one or more factors. These factors may either be ones which by their presence ensure expression of the gene coding for the cell function-limiting 25 enzyme or may, alternatively, be ones which suppress the expression of the gene so that their absence causes the enzyme to be expressed.

Factors regulating the activity of the promoter as defined above may be selected from a variety of factors. Thus, the 30 expression of the gene encoding the cell function-limiting enzyme may be determined by the environmental conditions or the physiological state of the cells, or by a cyclical or stochastic event. In the present context, the term "cyclical event" is understood to mean a cyclically recurrent event 35 causing changes in certain factors known to be potentially useful in influencing the expression of genes such as temperature conditions, changes in light intensity or hormonal changes. The term "physiological state of the ceils" denotes factors such as cell density or the growth phase of cells.

In accordance with the invention, advantageous promoter regulating factors are readily regulatable factors including the presence or absence of a certain chemical substance in the environment or the physical conditions in the environment such as the prevailing temperature or other physical factors 10 (e.g. the intensity of the light in the environment). Thus, it is possible to envisage containment systems as presently claimed, in which the gene coding for the cell function-limiting enzyme is expressed when a certain chemical substance present in a first environment such as the fermen-15 tation medium in which the cell is propagated, is not present in a second environment to which the cell is released, or when a factor required for the growth or survival of the cell is no longer present, or the factor is a factor which, when it is exhausted from an environment of the cell, has the 20 desired effect, viz. that the gene is expressed.

The promoter regulating the transcription of the gene coding for the cell function-limiting hydrolytically active enzyme may also become activated in a second environment of the cell by a chemical substance which is not present in a first 25 environment of the cell, but which is present in the second environment in sufficient quantities to activate the promoter. Similarly, the promoter may by a promoter which is activated by a shift in temperature, such as a shift from a higher temperature in a first environment as e.g. a fermen-30 tation vessel, to a lower temperature prevailing in an outside second environment, or the intensity of light, in that the promoter may be one which is activated in the presence of light of sufficient intensity, but is inactive in the darkness prevailing in a first environment such as a fermentation 35 vessel.

Dvo 95/10614 PCT/DK94/00381 21 Where cells as defined herein are ones that are to be released to the natural environment in a controlled manner, e.g. to a restricted area of land or to the intestinal tract of a human or an animal, the regulatable promoter may be one which 5 is regulated chemically, i.e. by the presence or absence of a certain chemical substance in -the environment of the cells as it has been explained above.

However, the regulatable promoter is advantageously a promoter which is activated cyclically, e.g. by changes of the 10 temperature, or most advantageously by a stochastic event. The term "stochastic event" as used herein is intended to denote an event which occurs at random at a certain frequency per cell per generation or frequency per unit time which, in accordance with the invention may result in a limitation of 15 the function of the cells in which the activation of expression of the cell function-limiting intracellularly active exoenzyme occurs, optionally to an extent which leads to the death of the cells. The stochastic event may be occasioned by periodic inversions of the region carrying the promoter, but 20 is more advantageously induced by the recombinational excision of a recombinationally excisable negatively functioning regulatory nucleotide sequence as defined above.

It should be noted that in order to ensure a general applicability of the present invention, the promoter used to ini-25 tiate transcription of the gene coding for the cell function-limiting enzyme is preferably a promoter which is capable of causing expression of said gene in a wide range of cells.

In case of regulatable transcription of the hydrolytically active enzyme, the regulatory nucleotide sequence may e.g. be 30 a promoter isolated from bacterial operons involved in the biosynthesis of amino acids or from bacterial genes, the transcription of which is activated late in the stationary growth phase or from bacterial genes involved in the synthesis of cell surface structures such as fimbriae. Examples 35 of suitable promoters are E. coli trp which becomes activated 22 in the absence of tryptophan, the bacteriophage X PR and PL promoters controlled by temperature sensitive regulatory nucleotide sequences, the Bacillus subtilis sporulation gene promoters which are activated during sporulation, and the E. coli and Salmonella fimbriae gene promoters which are activated stochastically.

In case of chemically regulatable promoters, the chemical substance, the presence or absence of which determines the activation of the promoter, may suitably be selected from 10 carbon or nitrogen sources, metabolites, amino acids, nucleosides, purine or pyrimidine bases or metal ions. When the chemical substance is one which, when present, suppresses promoter activity, it should preferably be a substance which rarely occurs in the natural environment in such concentra-15 tions that the promoter would not be activated when the cell is released to the natural environment. One example of a suitable promoter in e.g. an E. coli cell is the trp promoter which is repressed in the presence of tryptophan in the environment of the cell, but which is derepressed in the 20 absence of sufficient amounts of tryptophan in the environment. A containment system using the tip promoter or another promoter being regulated in the same manner, might therefore comprise an amount of tryptophan in a first environment, such as a fermentation vessel, to repress the promoter which is 25 derepressed when the cell is released from the first environment to a second environment, e.g. the natural environment which usually contains very low amounts of tryptophan or no tryptophan at all.

Another example of a regulatable promoter, the activation of 30 which is determined by a chemical substance is the lac promoter which is inducible by e.g. isopropyl-/3-D-thiogalacto-pyranoside (IPTG).

Where cells as defined herein are used in a pesticidally active composition e.g. being administered to a plant, the 23 regulatable promoter may suitably be regulated by the presence/absence of a compound secreted by this plant.

As mentioned above, the regulatable promoter may be a promoter, the activity of which is determined by the temperature 5 prevailing in the environment of a cell containing the gene coding for the cell function-limiting enzyme and a the regulatable promoter regulating the expression of the gene. In such a case, the regulation of the promoter is advantageously obtained by the presence in the cell of a temperature sensi-10 tive gene coding for a repressor for the promoter. As one typical example, the X promoters including those mentioned above may be regulated by a temperature sensitive X cl repressor .

Promoters which are activated stochastically by periodic 15 inversions of the promoter region (in the present context, such promoters are also termed as an "invertible promoter" and "inversional switch promoter") and which may be useful for the purposes of the present invention include as exanqples the hin, cin and gin promoters. One particularly useful 20 invertible promoter is the fimA promoter which is one E. coli fimbriae promoter. The activation (inversional switch) of this promoter is regulated by the gene products of the two genes which for the present purposes is termed the "on" and the "off" genes, the on gene product inducing a switch from 25 off (inactive) to on (active) , and the off gene product inducing a switch from on to off. In a wild-type E.coli cell where the fimA gene and its associated promoter is present in one copy on the chromosome, the inversional switch occurs with a switching frequency of about one cell/1000 cells/ 30 generation. It is, however, possible to regulate the frequency of the inversional switch as required by regulating the dosage of expression of the on and off genes. This may e.g. be effected by means of suitable promoters to transcribe into the on and off genes. The frequency of tramscription 35 initiation by these promoters will then determine the rela 24 tive dosage levels of the on and off gene products being formed.

As it has been explained above, the intracellular enzymatic activity of the cell function-limiting enzyme may also be 5 regulated by the presence or absence in the cell of a gene product rendering the cell refractory to said enzymatic activity, e.g. by interacting with the enzyme so that the cell function-limiting effect hereof is not expressed.

Stochastically induced regulation of the gene coding for the 10 hvdrolvticallv active exoenzvme bv means of recombinational exci3ion of negatively regulatory nucleotide sequences In accordance with the invention, one particularly advantageous method of stochastically regulating the expression of the gene coding for the cell function-limiting exoenzyme is 15 the induction of the gene expression as a result of recombinational excision of an excisable negatively functioning regulatory nucleotide sequence which, while present in the cell, inhibits expression of the gene. In the present context, the term "recombinational excision" refers to the 20 result of a naturally occurring phenomenon of genetic recombination (cross-over) whereby nucleotide sequences in repli-cons, in a controlled process, pair, brake and rejoin to form recombinant replicons by the sequential action of enzymes acting on the DNA. The frequency of recombinational events in 25 a cell depends i.a. on the degree of homology between paired complementary nucleotide sequences and on the length of the complementary sequences. Thus, it has been shown that about 50 base pairs of homology may be required to obtain recombination in a bacterial cell.

When a negatively regulatory nucleotide sequence is inserted between directly repeated nucleotide sequences of a sufficient length in a recombinationally proficient cell which, in accordance with the invention contains a gene coding for a cell function-limiting enzyme, recombination between the repeats results in the recombinational excision of the negatively regulatory nucleotide sequence allowing the gene to be expressed, whereby the cell funct ion -limiting enzyme may be produced intracellularly in amounts leading to a limitation 5 of the cell function, optionally resulting in the death of the cell.

Accordingly, the phenomenon of recombinational excision as used herein, implies that a DNA subsequence, i.e. the negatively regulatory nucleotide sequence, is excised from a 10 longer DNA sequence through a recombination event. In essence, the longer DNA sequence is cleaved on either side of the subsequence and the fresh ends are joined, leaving out the subsequence. Recombination occurs between sufficient homologous flanking nucleotide subsequences. Thus, with DNA 15 of the general structure W-X-Y-X-Z, X being a repeated sequence and Y being a negatively regulatory nucleotide sequence, this could recombine to form W-X-Z, with the Y subsequence being excised.

As mentioned above, the frequency of the recombination can to 20 some extent be determined by varying the lengths of the repeats and/or the distance between the repeats. Furthermore, the frequency may be varied by using repeat sequences of varying homologies. Thus, nucleotide sequence repeats being 100% homologous and having a size which does not impair 25 recombination will result in a high recombination frequency and hence, in a high frequency of recombinational excision of the negatively regulatory sequence, whereas mismatches within complementary sequences will reduce the recombination frequency depending on the degree of mismatch. As an example, it 30 has been found that 10% divergence between nucleotide sequence repeats may reduce the recombination frequency 40-fold.

Accordingly, the cell containing the gene coding for a hydrolytically active exoenzyme may, in accordance with the in-35 vention, be a cell containing a regulatory nucleotide se 26 quence which is a recombinationally excisable negatively functioning regulatory nucleotide sequence being flanked by a first flanking nucleotide sequence and a second flanking nucleotide sequence substantially homologous with the first 5 flanking sequence. As used herein, the term "substantially homologous with" is used to indicate that the degree of homology is sufficient to result in a desired frequency of recombination. In certain embodiments it may, in order to obtain a desirable maximum frequency of recombination, be 10 advantageous to use direct repeats, i.e. sequences being 100% homologous, whereas it may, in other embodiments where a moderate degree of cell function limitation is desirable, be appropriate to use repeats which are more or less heterologous, but still allowing a desirable lower frequency of 15 recombination to occur. Accordingly, in the present context, the term "sufficiently homologous" may appropriately be used to indicate a degree of homology between two flanking nucleotide sequence repeats which results in a desired frequency of recombinational events in a cell containing the gene coding 20 for the hydrolytically active exoenzyme and a negatively regulatory nucleotide sequence.

As it also has been mentioned above, the frequency of recombination depends on the lengths of the flanking sequences. In useful embodiments of the invention, flanking sequences are 25 used which have a length being in the range of 100-5000 base pairs. In certain preferred embodiments, it may be advantageous* to use flanking sequences, the length of which is in the range of 200-3000 base pairs. As the flanking sequences may be used any nucleotide repeats of sufficient lengths and 30 homology as it has been defined above. As one useful example of flanking sequences may be mentioned the chloramphenicol resistance gene having a size of about 900 base pairs and which occurs in the plasmid pBR325 (Bolivar, 1978, Gene, 4, 121-136). Another example of a useful nucleotide sequence 35 which when inserted as repeats may result in recombination, is a subsequence of the rmB gene isolated from the plasmid pKK3535 .(Brosius et al., 1981, Plasmid, £, 112-118) of the | WO 95/10614 PCT/DK94/00381 27 rrvB gene isolated from the plasmid pKK3535 (Brosius et al., 1981, Plasmid, £, 112-118) having a size e.g. in the range of 500 to about 3000 base pairs, such as 598 base pairs.

In one interesting embodiment of the invention, the cell 5 containing a gene whose expression in the cell results in the formation of a hydrolytically active enzyme is a cell wherein said gene is a gene which encodes a first RNA which is a messenger RNA, and which further contains an excisable negatively regulatory nucleotide sequence operably linked to said 10 gene encoding the hydrolytically active gene, which is a gene encoding a second RNA which forms an RNA-RNA duplex with said first messenger RNA and thereby, when it is expressed, inhibits translation of said gene coding for the hydrolytically active enzyme.

In another useful embodiment of the present invention, the recombinationally excisable negatively regulatory nucleotide sequence is a gene encoding a polypeptide repressor of transcription of the gene whose expression results in the intracellular formation of a hydrolytically active enzyme. Such a 20 polypeptide repressor may, e.g. be a lac repressor. As one specific example of a useful lac repressor may be mentioned the repressor encoded by the Laclq gene.

In a further useful embodiment of the invention, the excisable negatively regulatory nucleotide sequence is a trans -25 cription termination sequence, preventing the transcription of the gene whose expression results in the formation intracellularly of a hydrolytically active enzyme. In one specific embodiment of the invention, such a suitable terminator sequence may be the xrpoCt' transcription terminator isolated 30 from the plasmid pHBA102rpoCt (Squires et al., 1981, Nucleic Acid Res., 2., 6827-6839).

Negatively regulatory nucleotide sequences which in accordance with the invention are suitable, may be isolsted from nucleotide sequences derived from a virus, or a prokaryotic 28 or eucaryotic cell. Thus, sources of the nucleotide sequence include bacterial chromosomes, bacterial plasmids, prokaryotic viruses, eucaryotic viruses, eucaryotic plasmids, or eucaryotic chromosomes.

In preferred embodiments of the invention, the excisable negatively regulatory nucleotide sequence being operably linked to the gene coding for the hydrolytically active enzyme and the first and second flanking sequences, both as defined above, is provided in the form of a "cassette" which 10 term is used herein to describe a readily insertable nucleotide sequence comprising at least the above-mentioned sequences and optionally the gene coding for the hydrolytically active enzyme, and optionally further nucleotide sequences including as examples a suitable marker such as a gene coding 15 for antibiotic resistance. In the present context, the term "insertable" denotes that the cassette as defined herein is provided with suitable restriction sites at both ends allowing for insertion in a replicon having the same restriction sites. Accordingly, such preferred restriction sites include 20 sites which occur frequently in replicons where insertion is desirable or alternatively, restriction sites which may be easily provided in such replicons.

It will be understood that, in accordance with the invention, a cassette as defined above and which does not comprise the 25 gene coding for hydrolytically active enzyme and operably linked to the negatively regulatory nucleotide sequence, may be inserted in a replicon which is different from the replicon containing said gene. Optionally, the cassette as defined above may be inserted in a first replicon such as e.g. a 30 transposon and subsequently inserted via the transposon into the chromosome to obtain a cell as defined herein.

As it has been explained above, the activation of certain invertible promoters such as the fimA promoter or functional homologues hereof is regulated by the gene products of an on 35 gene and an off gene. It will be understood that this mechan WO f '10614 PCT/DK94/00381 29 ism of promoter regulation provides the possibility of using the off gene or a functional homologue hereof as a negatively reculatory nucleotide sequence which may be inserted in the cell as defined herein, as a recombinationally excisable 5 nucleotide sequence in the manner explained in details above. Accordingly, in one embodiment-, the present invention provides a cell wherein the gene whose expression results in the formation of a hydrolytically active enzyme is stochastically expressed as a result of recombinational inversion of an 10 invertible promoter sequence of the regulatory nucleotide sequence said promoter being operably linked to the gene.

Stochastically induced regulation of the cane coding for the hvdrolvticallv active exoenzvme bv means of site-specific recombinational excision of negatively regulatory nucleotide 15 sequences In plasmids, inherent mechanisms occur whereby multimer resolution of the plasmid during replication takes place. As exemplified by the broad host range plasmid RP4, this resolution system may comprise (1) a gene coding for a multimer 20 resolving enzyme, a resolvase and (2) a site for the site-specific resolvase-mediated resolution. In plasmid RP4 the gene coding for the resolvase is parA and the site for the resolution is designated mrs. If two mrs sites are placed in direct orientation, a nucleotide sequence inserted between 25 those two sites may, if the parA gene is present in the same host cell, be deleted at a relatively high frequency whereby a site-specific recombination system is provided. In useful embodiments the pax*A gene may be located in trans.

It has now been found that such a site-specific recombination 30 system provides a useful mechanism for stochastically regulating the expression of a gene such as the gene coding for the hydrolytically active enzyme as defined herein, since the site-specific recombination may be used to obtain recombinational excision of a negatively regulatory nucleotide 35 sequence as defined above.

Accordingly, in one interesting embodiment, the present invention provides a cell as defined herein in which the negatively regulatory nucleotide sequence is a sequence flanked by a first site for a site-specific resolution recom-5 binase and a second site for site-specific resolution, the second site being recognizable by the samr or a functionally equivalent multimer resolving enzyme as is the first site, whereby the regulatory sequence is recombinationally excisable in the cell. In a specific embodiment, the gene coding 10 for the multimer resolving enzyme is located in trans relative to the sites for site-specific resolution. In the present context, one useful example of a suitable gene is the parA gene isolated from plasmid RP4.

Provision of a cell according to the invention in which the 15 function-limiting effect of the hvdrolvticallv active enzvme is increased bv mutation In certain embodiments, it may be advantageous to obtain a higher intracellular enzymatic activity than is obtained by the insertion of the gene coding for the hydrolytically 20 active enzyme and/or the regulatory nucleotide sequence in the form in which this/these sequence(s) are primarily isolated. Such a higher activity may be the result of an increased aiuount of enzyme being expressed and/or of an increased specific activity of the enzyme, relative to the 25 amount or the specific activity which is obtained by the insertion of the primarily isolated sequences. An increased intracellular activity of the hydrolytically active enzyme may also be the result of an increased intracellular stability of the enzyme. Furthermore, a higher intracellular enzy-30 matic activity may be obtained with a modified enzyme which relative to a parent enzyme which to some extent is transported extracellularly, is retained essentially intracellularly.

Such increased enzymatic activity may conveniently be ob-35 tained by subjecting the isolated sequences separately or , WO 95/10614 PCT/DK94/00381 31 together to a conventional in vitro or in vivo mutagenization treatment e.g. with a chemical mutagen, by means of a site-directed mutagenesis, e.g. by use of a PCR technique, or by a mutagenically active radiation treatment.

Cells containing thus treated .nucleotide sequences in which a mutation or mutations resulting in an increased intracellular enzymatic activity have occurred, may be selected by growing a culture of the cells under conditions where the gene coding for the hydrolytically active enzyme is expressible, and 10 isolating cells or c«ll clones which relative to cells or cell clones containing the primarily isolated sequences and grown under the same conditions, show an increased level of cell function limitation as defined above, Accordingly, the invention providas in one embodiment a cell 15 as defined herein in which at least one of the gene whose expression results in the formation of a hydrolytically active enzyme and the nucleotide sequence regulating said gene, is mutated at one or more sites, whereby the cell function-limiting effect of the enzyme encoded by the gene, 20 when expressed in the cell, is the same or increased relative to the cell function-limiting effect of the enzyme expressed in a cell containing said gene and said nucleotide sequence in non-mutated form. In one interesting embodiment, the cell in which the enzyme activity is increased by the mutation 25 treatment is a cell containing a gene coding for a mutant nuclease encoded by a mutated Staphylococcus spp gene such as the nucleases encoded by pSNUC24-26 or pSNUC420-26 as described below.

A cell according to the invention comprising a further regu-30 latablv expressible gene which encodes a cell function-limiting gene product As a means of increasing the cell function-limiting effect in a cell as defined herein it may be advantageous to insert into the cell a further regulatably expressible gene which ^WO 95/10614 32 further gene is a gene encoding a non-enzyme cell function-limiting gene product or a further enzymatically active cell function-limiting gene product. Accordingly, the present invention provides in one specific embodiment a cell con-5 taining a further gene as defined above. Such a further regulatably expressible gene may be regulated by a regulatory nucleotide sequence of the same type as the sequence regulating the gene coding for the hydrclytically active enzyme or the gene may be one which is regulated by a regulatory 10 nucleotide sequence of another type, said other type of regulatory sequence optionally being one which is also capable of regulating the expression of the gene coding for cell function-limiting hydrolytically active enzyme.

A suitable gene coding for a further non-enzyme regulatably 15 expressible cell function-limiting gene product may conveniently be selected from the above-mentioned genes having cell killing function. In certain preferred embodiments, said further gene is selected from the hok gene of the parB region of plasmid Rl, the gef gene and a DNA sequence which is a 20 functional equivalent of either of these genes.

A suitable further enzymatically active cell function-limiting gene product is an exonucleolytically enzyme which is expressed intracellularly in the mature form including as au example the E. cold exonuclease III. One advantage of provides ing in a cell to be contained the simultaneous expression of the staphylococcal nuclease according to the invention and an exonuclease as defined above is that the enzymatic efficiency of the staphylococcal nuclease in the cell is enhanced.

A cell as defined herein which further contains a DNA se-30 ouence not naturally related to the replicon carrying the gene coding for the hvdrolvticallv active enzvme As it has been defined above, the cell according to the present invention may, when occurring as a population comprising a multiplicity of the cell which further comprises a WO 95/10614 PCT/DK94/00381 33 DNA sequence coding for an immunologically active enzyme, a pesticidally active or a pollutant-degrading gene product, respectively, be useful in an immunologically active composition, a pesticfdally active or a environmental pollutant-5 degrading composition.- respectively, to be contained in the particular environment where such a composition may be released.

Accordingly, the cell may in such specific embodiments be a cell which further comprises a DNA sequence not naturally 10 related to the replicon carrying the gene whose expression results in the formation of a hydrolytically active enzyme and/or the replicon carrying the regulatory nucleotide sequence, said DNA sequence being selected from a sequence coding for an immunologically active gene product, a sequence 15 coding for a pesticidally active gene product and a sequence coding for a pollutant degrading gene product.

In the present context, the term "immunologically active gene product" is used to describe an epitope (antigenic determinant) from a pathogenic organism which, when it is admini-20 stered to the body of a human or an animal is capable of stimulating the formation of antibodies therein. A cell as defined in the present invention which contains one or more genes encoding such a gene product may be utilized in the preparation of useful live vaccines. In the immunization 25 against several pathogens it is considered advantageous to administer live vaccines as compared to killed organisms or antigenic fragments of the pathogen, since the level of immunity conferred by a live vaccine is frequently higher than that conferred by vaccines comprising killed pathogenic 30 organisms or fragments thereof. Most known vaccines comprising viable epitope-containing organisms are either based on recombinant non-pathogenic organisms encoding the epitope or they are based on attenuated pathogenic organisms. The cell may advantageously contain a multiplicity of genes each of 35 which coding for a specific immunologically active gene product.

SUBSTITUTE SHEET WO 95/10614 PCIYDK94/00381 34 However, up till now the use of live vaccines has been limited since it is often difficult to obtain the right combination of attenuation, viability and adequate immune response. Furthermore, the deliberate release of genetically 5 engineered microorganisms to the body and to the external environment which is a result -of the use of viable recombinant:. organisms as vaccines, is currently not allowed in any country for reasons of public concern as to the possible long-term environmental impact, in particular the risk of 0 permanent establishment of the GEMs in the environment.

The present invention provides an advantageous means of circumventing these problems associated with the use of known GEM-based live vaccines by introducing into a viable epitope-containing cell the regulatably expressible gene coding a 5 cell function-limiting hydrolytically active enzyme as defined above. In particularly interesting embodiments, the invention provides as a useful basis for a viable vaccine, the cell as defined above which contains a gene coding for the hydrolytically active enzyme whose expression is sto-0 chastically induced.

In useful embodiments of the invention, the cell which contains the DNA sequence coding for an immunologically active gene product further comprises means for transporting the epitope, when expressed, to the outer surface of the cell, 5 i.e. translocating it across the cell membrane. Preferably such a translocation is obtained by inserting the gene coding for the epitope into a nucleotide sequence coding for an outer cell surface polypeptide structure such »,b fimbriae which contains the fimbrillin protein, pili, flagellae or 0 certain other surface proteins including as an example the OM protein found in Streptococcus species. By providing the cell with such a hybrid nucleotide sequence being expressible in the cell, the gene product hereof will be a fusion or hybrid protein comprising the epitope and the relevant cell surface 5 structure.

A cell in which a fusion protein is expressed which comprises the epitope fused to a surface structure protein by which the cell can adhere to the mucosal cells of a body to which the cell is administered is considered to be particularly useful 5 in that the epitope will become in close contact with the mucosa and thereby effectively, stimulate a protective immune response in the form of the excretion of secretory antibodies of the IgA and IgG classes.

Furthermore, the adhesion of the epitope-carrying cell will 10 ensure that the cell is retained in the human or animal body for a period of time which is sufficient to obtain the desired immune response. It is considered that a satisfactory immunization typically may be obtained if the cell is present in sufficient numbers in a particular body environment such 15 as the intestinal tract for a period in the range of 15-30 days, depending on the nature and the activity of the epitope expressed from the cell.

As it will be understood from the above description of the gene coding for the cell function-limiting hydrolytically 20 active enzyme and the regulatory nucleotide, the present invention may provide useful means of providing live vaccines based on recombinant organisms which are immunologically effective and which can be used without the risk of undesired spreading of recombinant genes to the microflora of humans 25 and animals or to the outer environment.

In accordance with the invention, a useful cell for the preparation of a live vaccine is one selected from a bacterial species which inherently contains an outer surface structure as mentioned above. Such species include as exam-30 pies species of Enterobacteriaceae such as Salmonella and E. coli species, Vibrionaceae and Pseudomonadaceae. It will be understood that strains of such species which are particularly useful in the present invention as the basis of a live vaccine as defined above, are non-pathogenic strains or 35 strains having a low pathogenicity. 36 The epitope expressed by a cell as defined above may be an epitope derived from any pathogenic organism or agent the obtainment of immunity against which is desirably. Such 5 pathogens include viruses, bacteria and eucaryotic organisms such as fungi, yeast or protozoa.

In commercially important embodiments, the cell may contain a nucleotide sequence coding for a pesticidally active gene product. In this context, the term "pesticidally active gene 10 product" is used to denote a product which when expressed in a cell being released to an environment where there is a need to reduce or eliminate the presence of pests including insect pests, vermins such as rodents cr birds. Such pests may be controlled by the administration of toxic chemical pesticides 15 to the infestated environment, but recently various naturally occurring pesticidally active organisms including viruses, bacteria and fungi have been used as biological pest control products.

Prominent examples of such pesticidally active organisms 20 include biotypes or strains of the species Bacillus thurxn-giensia which produce crystalline proteins being toxic to insects, in particular to caterpillars, and several viruses being pathogenic for insects in the larval stage or in the adult stage. However, the pesticidal effect of such organisms 25 is frequently less satisfactory and there is a strong need in farming, forestry and horticulture to provide improved pesticidally active organisms. One approach to solving this problem is to construct genetically engineered organisms having an increased toxic effect or a better survival rate in the 3 0 environment.

Should such improved organisms be developed, their use in the environment will, as a consequence of current public concern of the potential risks involved in deliberate release of such toxic or pathogenic GEMs, only be approved by official envi-35 ronmental agencies if it cam be demonstrated that the release 37 does not lead to an undesired propagation or to an extended survival of such organisms in the environment to which they are applied.

The present invention clearly provides the means of limiting 5 the survival in the environment of genetically engineered pesticidally active organisms. As it has been explained above, the rate of expression of the cell function-limiting hydrolytically active enzyme may be regulated stochastically and thus the survival rate of pesticidally active cells may 10 conveniently be adapted to any specific need. Also, the cell function-limiting effect may, in accordance with the present invention be adjusted by selecting a hydrolytically active enzyme which has an appropriate cell function-limiting effect.

In another useful embodiment, the invention provides a cell in which the DNA sequence not naturally related to the gene coding for the hydrolytically active enzyme, is a sequence coding for a pollutant-degrading gene product. It is known that several xenobiotic compounds polluting the outer en-20 vironment including soil and water can be degraded by microorganisms having an inherent capability of degrading these compounds. Obviously, the technology of genetic engineering provides means of providing improved organisms having an increased pollutant-degrading capacity or having the capacity 25 to degrade a broad range of compounds, in particular hydrocarbons .

However, the public concern as mentioned above are also relevant in this context and accordingly, the present invention provides useful means of providing improved pollu-30 tant-degrading cells, the survival of which can be controlled by regulating the expression of the cell function-limiting hydrolytically active enzyme as it is defined above. In particularly preferred embodiments, the cell contains a gene coding for a pollutant-degrading gene product, the expression WO 95/10614 PCT/DK94/00381 38 of which is induced by the presence of a pollutant degradable by the ceil.

Cell as defined herein which is a transformed cell In a further specific embodiment of the invention, the cell 5 according to the invention and as defined above, is a cell which is transformed with a recombinant replicon or recombinant replicons containing a gene whose expression results in the formation of the cell function-limiting enzyme, the expression of said gene being regulated by a regulatory 10 nucleotide sequence which is contained in the recombinant replicon containing the gene or in another recombinant replicon present in the transformed cell.

Replicon containing a gene coding for the intracellularly hvdrolvticallv active cell function-limiting enzvme and 15 optionally a regulatory nucleotide sequence As mentioned above, the present invention pertains in a further aspect to a recombinant replicon containing the regulatably expressible gene encoding an intracellularly active hydrolytic enzyme as defined above, having, when it is 20 expressed in a cell, a function-limiting effect thereon, said gene being regulated by a regulatory nucleotide sequence as also defined above and which sequence is operably linked to the expressible gene. In accordance with the invention, the regulatory nucleotide sequence may be a sequence contained in 25 the replicon or in another replicon present in the cell containing the expressible gene.

The replicon may in accordance with the invention also be a replicon wherein the gene coding for the hydrolytically active enzyme and/or the nucleotide sequence regulating the 30 expression of said gene, when such a sequence is present in the replicon, is mutated at one or more sites, so that the cell function-limiting effect of the enzyme encoded by the gene, when it is expressed in a cell containing the replicon, 39 is the same or increased relative to the cell function-limiting effect of the enzyme as expressed in a cell containing a replicon as defined herein not having been mutated.

In one embodiment, the replicon may comprise a further regu-5 latably expressible gene which encodes a non-enzyme cell function-limiting function, the expression of which may be regulated by a regulatory nucleotide sequence of the same type as the sequence regulating the gene coding for the cell function-limiting hydrolytically active enzyme. In other 0 embodiments, the nucleotide sequence regulating the expression of the non-enzyme cell function-limiting function is of a different type which however, may also be capable of regulating the expression of the gene encoding the cell function-limiting hydrolytically active enzyme.

Said further regulatably expressible gene may in useful embodiments be the hok gene from the parB region of plasmid R1 or a DNA sequence which is functionally homologous to the hok gene or the E. coli chromosomal gef gene.

In other useful embodiments, the replicon may further com-0 prise a DNA sequence as defined above which is not naturally related to the replicon and which encodes a gene product selected from an immunologically active gene product, a pesticidally active gene product and a pollutant-degrading gene product.

Population of cells containing a gene coding for an intracellularly hvdrolvticallv active exoenzyme having cell function-limiting effect As mentioned above, the present invention pertains in a further aspect to a population of cells consisting of a 0 multiplicity of cells as they have been defined above. The cell population may preferably comprise cell having been transformed with a recombinant replicon as defined herein which is capable of replicating in the cells. Since the cell 40 population in interesting uses may be released to the outer environment, the cells may in advantageous embodiments be bacterial cells which are selected from species whose natural habitat is a habitat selected from soil, surface water and 5 plants, such as gram-negative bacterial species.

In this context, it is interesting to note that the survival of a population of cells as presently defined and in which the expression of the gene coding for the cell function-limiting enzyme is regulatable by a repressor substance which 10 is present in the cells, but in different amounts depending on the physiological state of the cells, may, when such a population is applied to a human or an animal body, or to the outer environment, be regulated as a result of a decay of the repressor substance to an extent whereby the repressor sub-15 stance is converted to a non-functional form. Provided the amount of the repressor substance in individual cells of a population being released, is different, it is assumed t .aat this decay will result in a gradually increasing loss of viability of the cells of the population.

Method of limiting the survival of a cell population As it has also been mentioned above, the present invention provides in one aspect a method of limiting the survival of a cell population in a first or a second environment wherein the regulatory nucleotide sequence is regulatable by an 25 environmental factor as defined herein. The method may in preferred embodiments be related to a cell population as defined above.

In specific embodiments of this method, the survival of the cell population is limited in a first environment in which 30 the gene coding for the cell function-limiting hydrolytically active enzyme is expressed whereby the cell population is contained in the first environment. In the present context, a first environment is typically the place of primary propagation of the cell such as a fermentation vessel.

WO 95/10614 PCI7DK94/00381 41 In another specific embodiment of the method, the survival of the cell population is limited in a second environment which may be a first environment changing to a chemically different second environment, e.g. by the depletion of certain chemi-5 cals or the addition of such chemicals, or to a physically different second environment which change e.g. may take place by a shift in temperature or by a change of light intensity.

As it has been mentioned above, a cell population as defined herein may also by a cell population wherein the expression 10 of the gene coding for the function-limiting enzyme is regulatable by a repressor substance which can undergo a decay or a degradation when said cells are released to a human or animal body or the outer environment to an extent whereby the repressor substance is converted to a non-functional form, 15 said repressor substance being present in the cells of the population in different amounts whereby as a result of said decay the function of the cells of the population will gradually be limited.

Accordingly, the present invention provides in one useful 20 embodiment a method of limiting the survival of a cell population by providing the cells with a gene coding for the hydrolytically active enzyme which is operably linked to a regulatory nucleotide sequence encoding a repressor substance which can undergo a decay when said cells are released to a 25 human or animal body or the outer environment, to an extent whereby the repressor substance is converted to a non-functional form, said repressor substance being present in the cells of the population in different amounts whereby as a result of said decay the function of the cells of the popula-30 tion will be gradually be limited.

WO 95/10614 PCT/DK94/00381 42 A method of containing an extrachromosomal recombinant replicon As defined above, the invention relates in one aspect to a method of containing an extrachromosomal recombinant replicon 5 to a first kind of cells where, the replicon is naturally transferable to a second kind of cells, in which method the recombinant replicon is preferably a recombinant replicon as defined herein. In one preferred embodiment, this replicon is one not containing a regulatory nucleotide sequence and 10 advantageously, such a replicon contains a gene coding for a hydrolytically active enzyme which is an endonuclease capable of hydrolysing diesterphosphate bonds in nucleic acids in the first kind of cells containing the replicon to be contained.

Method of stochastically limiting the function of cells in a is cell population In a particularly interesting aspect, the invention pertains, as it has been mentioned above, to a method of stochastically limiting the survival of a cell population such as a cell population as defined above, said method comprising the 20 transformation of the cells with a recombinant replicon as also defined herein. In one useful embodiment, the method comprises a method wherein the expression of the cell function-limiting hydrolytically active enzyme is induced as a result of a site-specific recombinational excision of an 25 excisable negatively functioning regulatory nucleotide sequence which, while present in the cells, inhibits expression of said gene, the negatively functioning regulatory nucleotide sequence being contained in the recombinant replicon or in another recombinant replicon present in the cells.

In another useful embodiment of this method, the survival of the cell population transformed with a recombinant replicon as presently defined, is being limited as a result of the above-defined recombinational inversion of an invertible promoter sequence of the negatively functioning regulatory 43 sequence. In accordance with the invention, the negatively functioning regulatory sequence may be one contained in the recombinant replicon or it may be one present on another replicon present in the cells. As one suitable example, the 5 promoter sequence may be a sequence carrying the fimA promoter or a functional homologue thereof.

An immunologically active composition The immunologically active composition as defined above may in addition to the viable function-limited cell population 0 comprise pharmaceutically acceptable carriers and additives. Such acceptable carriers include any vehicle which is used conventionally in vaccine production such as e.g. saline. In the present context, suitable additives include immune response-enhancing substances including as examples Freund's 5 incomplete and complete adjuvant and other non-specific immunostimulating substances.

It may be preferred to provide the composition in the form of lyophilized compositions, optionally in combination with a suitable aqueous vehicle such as saline. The immunologically 0 active composition as presently defined may contain different types of cells, each of which encoding a specific epitope. Alternative, the immunologically active composition may in accordance with the invention contain a population of cells containing a multiplicity of genes coding for an immunologi-5 cally active gene product, each gene coding for a different gene.product.

The immunologically active compositions provided herein are useful vaccines for the immunization of both humans and animals. 0 In preferred embodiments, the immunologically active composition contains cells containing a sequence encoding for an immunologically active gene product w»*ich is a sequence coding for a fusion protein comprising said gene product and 44 a polypeptide, the presence of which results in the transportation of the fusion protein to the outer surface of the cells. As suitable examples, the composition may contain cells wherein the polypeptide is one selected from a polypep-5 tide selected from a bacterial fimbrillin protein, a bacterial pilus, a bacterial flagellum and a bacterial OM surface protein.

Nucleotide sequences coding for such a polypeptide may conveniently be isolated from a bacterium selected from Entero-0 bacteriaceae, Vibrionaceae and Pseudomonadaceae.

A pesticidally active composition A pesticidally active composition as defined herein may in accordance with the invention, in specific useful embodiments contain a further DNA sequence encode a gene product which is 5 toxic for insects or their progeny. In one interesting embodiment, the further DNA sequence is one derived from a strain of Bacillus thuringiensis which codes for an insec-ticidal protein.

As it has been explained above, cells as defined herein may 0 code for a variety of pesticidally active gene products including the insecticidal protein mentioned above. Accordingly, this composition may be useful in controlling a wide range of pests and vermins. Cells which are useful in the pesticidally active composition may suitably contain a gene 5 coding for the active gene product which are isolated from naturally occurring organisms producing or having a pestici-dal activity.

The pesticidally active gene products coded for by the cells of the composition may be gene products which have a toxic 0 effect on a pest or vermin, or the gene products may be a pest pathogenic virus which are expressed in the cells of the composition. The cells which in accordance with the present invention may be used in this composition include cells in 45 PCX /DK94/00381 which the gene coding for a desired pesticidally active gene product can be expressed. Such cells include bacterial and fungal cells and plant and aniinal cells, optionally grown as cell cultures.

The pesticidally active composition may further comprise a suitable carrier. In this context, the term "suitable carrier" is used to indicate that the carrier comprises compounds which enhance the spreading of the composition in the pest infestated environment such as bulking agents which do 0 not limit the survival of the cells in the composition, and compounds which may ensure the maintenance of the viability of the cells during production and storage of the composition and optionally also after application to the environment. As an example, a carrier may contain a compound which protect 5 against the ultraviolet light which is detrimental to many organisms.

An environmental pollutant-degrading composition The environmental pollutant-degrading composition as defined herein may preferably contain cells wherein the gene coding 0 for the cell function-limiting hydrolytically active enzyme is only expressed when the pollutant degradable by the pollutant-degrading gene product is substantially degraded. As sin example, such a composition may contain cells in which the expression of the hydrolytically active enzyme is repressed 5 in the presence of the degradable pollutant.

DESCRIPTION OF THE DRAWINGS Fig. l shows the nucleotide sequence of the Staphylococcus aureus (Foggi) nuclease including the signal peptide encoding sequence, and the amino acid sequence of the signal peptide and the mature enzyme (SEQ ID NO:5, SEQ ID NO:6), ^ WO 95/10614 46 Fig. 2 shows the construction of pSNUC24-26 by ligation of PCR amplified, truncated Staphylococcus aureus (Foggi) nuclease encoding gene fragment digested with BamHI and SacI (deletion of 9 amino acid residues of the mature nuclease 5 protein) to pET24d(+) also digested with BanHI and SacI, Fig. 3 shows the nucleotide sequence of the mutant staphylococcal nuclease gene in pSNUC24-26 (SEQ ID NO:7). Amino acids in boxes represent essential amino acids required for binding to Ca2+ providing the conformational stability of the pro-10 tein. Amino acids which are doubly underlined are important for conformational stability of the protein. A * indicates amino acid residues after mutation. Amino acid positions in brackets represent the position of the amino acids in the wild type nuclease gene without signal sequence (mature 15 protein), Fig. 4 shows kinetics of staphylococcus nuclease (SNUC) induction in E. coli HMS174(DE3) harbouring pSNUC24-26, Fig. 5 illustrates intracellular stability of the mutant staphylococcal nuclease encoded by pSNUC24-26, Fig. 6 shows kinetics of staphylococcus nuclease (SNUC) induction in E. coli NM522 harbouring pSNUC420-26, and Fig. 7 illustrates intracellular stability of the mutant staphylococcal nuclease encoded by pSNUC420-26.

EXAMPLES The below reference examples 1 and 2 corresponds to Examples 13 and 14, respectively in the co-pending application WO 93/20211 and are included herein for reference purposes. In these reference examples indications of figures refer to figures in the above co-pending application.

REFERENCE EXAMPLE 1 47 IfrtraceilMay expression and development of » copflitAoaaJ- cell function-limiting system in Escherichia coli usincr a nuclease gene from Staphylococcus aureus A. Bacterial strains, plasmids and oligonucleotides used E. coli JM109 was purchased from Invitrogen Inc., San Diego, CA. Plasmid pFOG 408 containing the nuclease gene of Staphylococcus aureus Foggi strain was a gift from A. Meeker. The cloning vector plasmid pUHE24-2 was obtained from S. Molin. 0 The oligonucleotides for PCR amplification were custom-synthesized by GENOSYS Inc., USA.

B. PCR amplification of the nuclease gene The nuclease gene from the plasmid pFOG 408 without its signal sequence was PCR amplified using two oligonucleotide 5 primers L-SNUC and R-SNUC. The primer L-SNUC (SEQ ID NO:l) was 37 nucleotides long (5' GATCCGGATCCGCAACTTCAAC-TAAAAAATTACATAA-3') with a 11 nucleotide overhang at the 5'-end consisting of flamHI, AccII, and Jfpall restriction endonuclease sites. The primer R-SNUC (SEQ ID NO:2) was 33 0 nucleotides long (5' -GGTACCGGAATTCGTGCCACTAGCAGCAGTGAC- 3') with EcoRI, ffpal, and Kpnl restriction endonuclease sites. PCR amplifications were performed in a DNA thermal cycler 480 (Perkin Elmer Cetus) using AmpliTag DNA polymerase (2.5 units) (Perkin Elmer Cetus) , 200 piM of each of the dNTPs 5 (Pharmacia), PCR reaction buffer (10X reaction buffer contained 500 mM Tris.HCl [pH 8.9], 500 mM KC1, and 25 mM MGC12), and 0.5 /xM of each of the primers. The PCR amplification was performed for a total of 25 cycles, each cycle consisting of 1 min denaturation at 94°C, l min primer annea-0 ling of 55°C, and 1 min primer extension at 72 °C. Successful PCR anplification of a 0.507 kb DNA fragment was determined 48 by running 1% Seakem agarose gel (1 AC Bioproducts), stained with EtBr, and visualized under a OV transilluminator.

PCR amplification using the primers L-SNUC and R-SNUC produced a single DNA band of 0.507 kb in size indicating no 5 non-specific priming. The upstream primer L-SNUC was designed precisely so that it would prime the first nucleotide of the first amino acid residue after the signal sequence of the gene (cf. the sequence defined above). Moreover, a 11 bp overhang at the 5'-end of the primer ensured the synthesis of 10 the gene from the desired nucleotide; thus eliminating the possibility of frameshift mutation during PCR amplification. The downstream primer R-SNUC was located further downstream of the stop codon and the possible hairpin structure outside the coding sequence of the gene, ensuring that all the amino 15 acid including the stop codon of the gene remain intact and functional.

C. Cloning of the amplified nuclease gene The amplified DNA of the staphylococcal nuclease gene without its signal sequence wa«j end-repaired with DNA poll Klenow 20 fragment to create blunt ends as described by Ausubel et (1987). The end-repaired blunt ended amplified nuclease gene was purified by using a Centricon 100 microconcentrator (Amicon, MA) . pUHE24-2 was linearized with BamHl, end-re-paired to create blunt-ends and purified by following the 25 procedures described by Ausubel et al (1987). Cloning and t-.rans format ion of the cloned DNA into E. coli JM109 was performed as described by Ausubel et al (1987). The transformed colonies were screened for the appropriate clones by replica plating on DNAse test agar (Difcc) plates containing 30 methyl green as indicator dye, 40 fig ampicillin per ml, 1 mM Isopropyl-S-D-thiogalactopyranoside (IPTG), 10 mM CaCl2, and 1 mM MgCl2.

Colonies which produced indicator colour were further tested for clones by restriction analysis, PCR amplification, and 49 DNA sequence analysis. Plasmid DNAs from the putative clones, designated as pSNUC-1 and pSNUC-3 were isolated and purified by alkaline lysis method as described by Ausubel et al (1987) . The isolated plasmid DNAs were digested with Hixadlll 5 restriction enzyme (US Biochemical??) according to the manufacturer. The restriction enzyme digested plasmid DNAs were analyzed by 10% polyacrylamide gel electrophoresis, stained with EtBr, and visualized by a UV transilluminator. The nucleotide sequence analysis of the cloned fragments in both 0 plasmids, pSNUC-1 and pSNUC-3, were determined by Sanger dideoxy method (Sanger et al, 1977) using a Sequenase sequencing kit (US BiochemicaIs) and the L-SNUC primer. These two clones were further characterized for induction and expression of the nuclease. Also they were tested for their ability 5 to grow on LB agar plates containing ampicillin (40 /zg per ml) , 10 mM CaCl2, 1 mM MgCl2, with or without 1 mM IPTG.

The transformed cells were initially selected on LB agar plates containing 40 /xg per ml ampicillin. Two putative clones which showed indicator colour change on DNAse test 0 agar plate, were further tested on LB agar containing ampicillin, CaCl2, MgCl2, and IPTG. E. coli JM109 containing pSNUC-1 showed no growth on agar plate containing IPTG even after incubation up to 72 hours (Fig. 11). In contrast to this, E. coli JM109 carrying pSNUC-3 showed inhibited growth 5 on similar type of agar plate containing IPTG (Fig. 12). In both cases, the control LB agar plates containing CaCl2 and MgCl2, but no IPTG showed complete growth within an 18-24 hours' time period. In this experiment, the inoculum sizes were kept as equal as possible for both types of plates, and 0 they showed consistent results every time when repeated for a total of 7 times.

Restriction analysis of the plasmids pSNUC-1 and pSNUC-3 with Hindlll produced expected DNA bands of approximately 0.219 kb and 4.0 kb bands (Fig. 13). Since there was one Hindlll 5 restriction site within the coding sequence of the nuclease WO 95/10614 PCT/DK94/00381 50 gene and one u:, the vector, it was possible to determine the right orientation of the nuclease gene on the vector.

Nucleotide sequence analysis of the first 40 bases of the cloned fragments in both pSNUC-1 and pSNUC-3 plasmids at 5 their 5'-ends confirmed the presence of the nuclease gene in the right orientation.

D. Induction of pSNUC-1 and pSNUC-3 The E. coli JM109 (pSNUC-1) and E. coli JM109 (pSNUC-3) were grown in LB broth containing 10 mM CaCl2 and 1 mM MgCl2 at 10 37°C for l hour with shaking at 100 rpm till the optical density (OD450) reached 0.5-0.7 when 1 mM sterile IPTG solution was added ior induction. Before and after the addition of IPTG the OD450 was recorded every hour, a fraction of the cultures was serially diluted in phosphate buffer (pH 7.2) 15 and plated on LB agar plates with 10 mM CaCl2 and 1 mM MgCl2 to determine viable plate counts. A control culture of each of the plasmid constructs was kept to compare the expression and killing efficiencies of the nuclease gene.

E. Expression of the cloned nuclease gene following induction E. coli JM109 (pSNUC-1) and E. coli JM109 (pSNUC-3) were grown in LB broth as described earlier to mid-exponential phase (OD450 of 0.4 to 0.5) and IPTG was added for Induction. The induction was carried on for 20 min when the cells were centrifuged and prepared for the total protein analysis by 25 SDS - PAGE as described by Ausubel et al (1987) using the Mini-Protean II gel system (BioRad). The PAGE separated proteins were stained with commassie blue. Uniaduced cultures of E. coli JM108 (pSNUC-1) and (pSNUC-3), cultures of E. coli AR120 (pFOG408) , and E. coli JM109 (pUHE24-2) were used for con-30 trols.

For further characterization of the cloned nuclease genes in pSNUC-1 and pSNUC-3, a western blot was performed using 51 rabbit antibody (a gift from A. Meeker). The proteins from the PAGE were transferred to nitrocellulose membrane (Mil-lipore) using the Mini-Trans Blot system (BioRad). A 1:15,000 dilution of rabbit antibody was used and detected with bio-5 tinylated. goat antirabbit immunoglobulin and streptavidin peroxidase (Fisher Biotech). The color development was performed by incubating in the peroxidase substrate by 4-chloro-l-naphthol. Appropriate controls were tested in this experiment.

Induction of E. coll JM109 (pSNUC-1) with IPTG showed significant decline in cell numbers between 2-7 hours' time period as determined by the OD450 reading and viable plate counts (Figs. 14a and 14b). The total cell number for E. coll JM109 (pSNUC-1) per ml declined form 2xl06 to 1.5xl04 between 0-7 15 hours' time period as determined by viable plate counts. In contrast to this, when E. coll JM109 (pSNUC-3) was induced with IPTG, a slow increase in cell number was evidenced as compared to the uninduced control as determined by the OD450 measurements and viable plate counts (Figs. 15a and 15b). The 20 total cell number increased form 3xl06 to 7x10® per ml between 0-7 hours as compared to the uninduced control which was 3xl06 to 2xl010 within the same time period. From these results it can be predicted that the lower killing efficiency of the plasmid pSNUC-3 upon induction as compared to pSNUC-1 25 may be due to mutation generated by misincorporation of nucleotide(s) during the PCR DNA amplification process. However, it is not obvious which of the two clones, if not both, has the altered nucleotide sequence.

The phenotypic expression of the cloned nuclease genes in 30 pSNUC-1 and pSNUC-3 plasmids on the DNAse test agar containing methyl green indicator dye showed expected color change. The pSNUC-1 showed more rapid color change than pSNUC-3. The control plasmid vector pSM1128 did not show any change in color on the same agar plates, whereas, plasmid pFC)G408 35 showed significant color change. 52 The PAGE gel for total protein analysis showed faint bands of approximately 17.8 kD size range for plasmids pSNUC-1 and pSNUC-3 following induction with IPTG. The uninduced controls and pUHE24-2 did not show any such band (Fig. 16) . Since 5 nuclease is an extracellular secretory protein, the E. coli strain carrying pFOG4()8 plasmid showed a very faint band in the gel. To confirm the expression of the cloned nuclease gene in plasmids pSNUC-1 and pSNUC-3, a western blot was performed. In the western blot, distinct protein bands of 10 approximately 17.8 kD were evidenced from the induced E. coli JM109 strains carrying pSNUC-1 and pSNUC-3 plasmids, and E. coli AR120 strain carrying pFOG408 plasmid (Fig. 17). The uninduced cultures and control plasmid pSM1128 did not show any band. Another non-specific band of approximately 20 kD 15 size was noticed in all samples, which seems to be the nonspecific antigenic reaction against the secondary antibody. This experiment suggested that the nuclease was produced by both plasmids pSNUC-1 and pSNUC-3 upon induction. The lower killing efficiency for the pSNUC-3 plasmid could be due to 20 the altered nucleotide sequence which has chcinged the protein structure and its functions for the substrate. Alternatively, this could be the case for the plasmid pSNUC-l for its relatively higher killing efficiency. Complete nucleotide sequence analysis for both clones may resolve this question 25 and also provide the information on possible sites where change of nucleotide sequence may make this enzyme more effective. 53 REFERENCE EXAMPLE 2 ' Determination of intracellular stability of cloned staphylococcal nuclease in Escherichia coli bv ELXSA assay The staphylococcal nuclease gene snuc without its signal 5 sequence was PCR-amplified and cloned in Escherichia coli JM109 as described in Exanqple 13 using the expression vector pUHE24-2, in the BamHI restriction site (pSNUC-l and pSNUC-3). The nuclease was expressed intracellularly when induced with IPTG. The stability of the intracellularly expressed 10 SNUC enzyme in E. coli following induction was determined by ELISA assay using antibodies raised in rabbits. Two approaches were pursued to determine the stability of the intracellularly expressed SNUC enzyme: 1. Transcriptional inhibition following induction by blocking 15 mRNA synthesis with rifampicin. 2. Removal of inducing agent (IPTG) from the culture following induction. 1. Transcriptional inhibition following induction bv blocking mRNA synthesis with rifampicin E. coli cells harbouring pSNUC-1 or pSNUC-3 were grown in liquid broth at 37°C till the optical density (OD450) was between 0.12 and 0.15 (early exponential growth phase). The cells were then induced with IPTG for 30 minutes, and rifampicin was added to the culture to block tramscription. The 25 incubation was carried on for the next 3 hours. E. coli JM109 without pSNUC-1 or pSNUC-3 was used as the control.

Aliquots of l ml of the cultures were collected before induction, 30 minutes after induction, and every 30 minutes after the addition of rifampicin. The cells were centrifuged, 30 washed twice with phosphate buffer (pH 7.2), and disrupted by WO 95/10614 PCT/DK94/00381 54 sonication to release the total cellular proteins. The soni-cation was carried on (8 to 10 minutes with a pulse for 30 seconds at every 30 seconds interval) till the sample, became clear. The debris was spun down and the supernatant (lysate) 5 was used for ELISA assay.

An aliquot of each of the samples was subjected to spectrophotometry measurement to determine the total protein concentration. ELISA assay was performed using equal quantity of total cellular protein from each lysate. Typically, 10 to 55 fil of lysates (depending on the concentration of each lysate) from various samples were absorbed in a microtiter plate (Corning) for about 16 hours (overnight) at 4°C. The wells were then washed 5 times with phosphate buffer (pH 7.2). The unreacted surface of the microtiter plate was blocked with 2% bovine serum albumin (BSA) from Sigma Chemical at room temperature for 1 hour with gentle shaking. The excess BSA from the microtiter plate was washed off 5 times with phosphate buffer (pH 7.2). 1:15.000 fold diluted polyclonal primary antibodies raised against the purified extracellular staphylococcal nuclease in rabbits, was reacted with pi^absorbed total cellular proteins for about 16 hours (overnight) at 4°C. The unreacted primary antibodies were washed off 5 times with phosphate buffer (pH 7.2).

The bound primary antibodies were then treated with secondary antibodies, goat antirabbit-Ig-biotin (CloneTech) for l hour at room temperature with gentle shaking. After washing off excess secondary antibodies, streptavidin-horseradish peroxidase (HRP-SA) conjugate was added to the microtiter plate and conjugated with the secondary antibodies. After washing off excess HRP-SA, colour development was carried out using 0.1 ml of substrate [2,2'-azinobis (3-ethyl benzthiazoline sul-phonic acid) in a 0.1 M citrate buffer, pH 4.2-with 0.03% hydrogen peroxide]. The absorbance was read at 415 nm in an ELISA microtiter plate reader (Flow Laboratories, Inc., McClean, Virginia). 55 PCTZDK94/00381 2. Removal of inducible agent (IPTG) from the culture following a short induction stage E. coli cells harbouring pSNUC-l or pSNUC-3 were grown to early exponential phase (OD450 of 0.12 to 0.15) when 1 mM 5 IPTG (final concentration) was. added to the culture. The induction with IPTG was carried on for 5 minutes at 37°C and the cells were centrifuged and washed twice with phosphate buffer (pH 7.2). The cells were resuspended in broth and incubated at 37°C for another 3 hours. Aliquots of 1 ml each 10 of the cultures were collected before induction and 5 minutes after induction. Also, aliquots were collected by centrifuga-tion, washed twice with phosphate buffer (pH 7.2) and sonicated to release total cellular proteins. Cultures of E. coli JM109 without pSNUC-1 or pSNUC-3 was used as the negative 15 control.

Total cellular proteins were released by disrupting the cells using a sonic disruptor as described above. ELISA assays for intracellularly expressed staphylococcal nuclease was also performed as described above.

The amount of staphylococcal nuclease protein encoded by pSNUC-1 and pSNUC-3 after transcriptional inhibition as defined above, was found to be significant amounts following induction with IPTG. The data for pSNUC-1 and pSNUC-3 are summarized in Tables 5 and 6, respectively. The expression of 25 staphylococcal nuclease increased about 2-fold during the first 30 minutes after induction as compared with the amount detected prior to induction. The activity of the nuclease enzyme remained during the first 2 hours following rifampicin treatment. Between 2 and 3 hours after addition of rifampicin 30 (2.5 and 3.5 hours after induction with IPTG) the activity of the nuclease was dropped significantly. From this experiment it can be concluded that the staphylococcal nuclease is stable intracellularly for at least 1.5 hours after the inhibition of transcription. 56 Table 5. Determination of the stability of intracellularly expressed staphylococcal nuclease expressed by pSNUC-1 following induction with IPTG and treatment with rifampicin Treatment Time (h) ELISA readings 0 0.427 IPTG 0.5 0.584 Rifampicin 1.0 0.666 1.5 0.685 2.0 0.675 2.5 0.542 3.0 0.488 3.5 0.407 Negative control 1.0 0.276 Table 6. Determination of the stability of intracellularly 20 expressed staphylococcal nuclease expressed bv pSNUC-3 following induction with IPTG and treatment with rifampicin Treatment Time (h) ELISA readings 0 0.399 IPTG 0.5 0.495 Rifampicin 1.0 0.557 1.5 0.593 2.0 0.555 2.5 0.443 3.0 0.427 3.5 0.378 Negative control 1.0 0.258 57 After induction of with IPTG for a short period of time (5 minutes), the cells were centrifuged, washed with phosphate buffer (pH 7.2),. and grown in a rich medium for 150 minutes to determine the stability of the SNUC in the cells.

The summary of the data is presented in Tables 7 and 8. There was at least 3-fold increase in the amount of SNUC following induction. The SNUC protein was found to be stable in pSNUC-l for 1.5 to 2 hours (between 5 and 125 minutes) after induction and l to 1.5 hours (between 5 and 95 minutes) in pSNUC-3 10 as it was determined by the ELISA assay. After 125 minutes for pSNUC-l and 95 minutes for pSNUC-3 following removal of the inducing agent (IPTG), the SNUC activity decreased significantly.

Table 7. Determination of the stability of intracellularly 15 expressed staphylococcal nuclease expressed bv pSNUC-1 following removal of IPTG after 5 minutes of induction Treatment Time (min) ELISA readings 0 0.270 IPTG 0.565 Removal of IPTG 65 95 125 155 0.560 0.543 0.501 0.456 0.329 Negative control 0.204 WO 95/10614 PCT/DK94/00381 58 Table 8. Determination of the stability of intracellularly expressed staphylococcal nuclease expressed bv pSNUC-3 following removal of IPTG after 5 minutes of induction Treatment Time (min) ELISA readings IPTG 0.171 0.461 0 Removal of IPTG 0.458 65 0.430 95 0.375 125 0.269 155 0.210 Negative control 35 0.164 EXAMPLE 1 Intracellular expression and stability of a mutant Staphylococcus aureus nuclease gene in Escherichia, coli A. Cloning of the mutant staphylococcal nuclease gene for intracellular expression In applicants' co-pending application US Serial No. 08/135,665 it is disclosed that the wild type Staphylococcus aureus (Foggi) nuclease gene isolated from the plasmid pFOG408, without its signal sequence may be expressed intracellularly and used as the basis for a conditional cell function-limiting system in bacteria. However, it has been found that although the expression of the wild type nuclease protein is predominantly intracellularly, some export of the signal peptide-less enzyme outside the cell may be observed. Accordingly, it was attempted to prevent such extracellular 59 transport of staphylococcal nuclease by removal, not only of the signal peptide, but of further amino acid residues of the mature protein.

DNA sequences encoding a total of 30 amino acid residues (21 5 amino acid residues of the signal peptide sequence and 9 amino acid residues of the mature protein, cf. Fig. 1) of the nuclease derived from the Staphylococcus aureus (Foggi) strain (ATCC 27355) were deleted and the rest of the gene segment constituting 426 bp was PCR amplified using oligonu-0 cleotide primers flanked with appropriate restriction enzyme sites and cloned into pET24d(+) plasmid vector (5307 bp) (Novagen, Inc. 597 Science Drive, Madison, WI 53711). The location, lengths and the nucleotide sequences of the oligonucleotide primers which were used for PCR amplification of 5 the staphylococcal nuclease gene are described in Fig. 3. For the intracellular biological activity, the specific amino acid residue # 31 (Glu encoded by the nucleotide sequence GAA) at the N-terminal end of the nuclease gene was selected as the first amino acid of the gene. This selection of the 0 first amino acid was based on Tucker et al., 1979, Molecular and Cellular Biochemistry, 22., 131-141, according to which the deletion of amino acid residues starting from # 10 and onwards of the Staphylococcus aureus (Foggi) nuclease produces a marked fall in both structure and activity of the 5 enzyme.

For cloning, the pET24d(+) vector was treated with BamHI and SacI restriction endonucleases and ligated to similarly digested PCR amplified staphylococcal nuclease gene fragment as defined above. The pET24d(+) plasmid vector comprises a 0 promoter sequence from bacteriophage T7, the lac operator (lacO) DNA segment which interacts with the repressor protein in the absence of IPTG, a DNA sequence for ribosome binding site (rbs), followed by several unique bacterial restriction endonuclease sites (multiple cloning sites or MCSs) for the 5 insertion of a foreign DNA segment. The repressor protein is encoded by the lacl gene which is located on the pET24d(+) WO 95/10614 FCT/DK94/00381 60 vector. Also, the pET24d(+) vector comprises a gene that encodes for resistance to kanamycin (KM) , and the colEi replication origin. The DNA sequence (ATG) that codes for the translational start codon (Met) is located immediately after 5 the rbs in the multiple cloning site for expression of the nuclease protein from the truncated nuclease gene segment without the translational first codon. There was no BaxriSl or SacI restriction endonuclease sites within the nuclease gene fragment.

The pET24d(+) vector ligated to the digested PCR amplified staphylococcal gene fragments as defined above was used for transformation of the E. coli strain HMS174(DE3) (genotype: F", recA, rkl2" > mki2"' Rifr) (Novagen, Inc., Madison, WI) which carries the T7 RNA polymerase gene tinder the lacUV5 15 control located on the chromosome. Transformants were selected on DNAse agar with toluidine blue (BBL cat# 99081) supplemented with 75 fig per ml of kanamycin. A number of clones with the characteristic nuclease "halo" were identified after 16-24 h of incubation at 37°C. However, a few 20 colonies were found to be relatively slow growing on the same agar plates and showed secondary nuclease activity as evidenced by a relatively slowly developing "halo" on the DNAse test agar. One of these relatively slow growing colonies was selected and further tested with regard to the nature of the 25 nuclease gene. The cloned nuclease gene in the pET24d(+) which was isolated from the slow growing E. coli HMS174(DE3) transformants was designated as pSNUC24-26 (Fig. 2). pSNUC24-26 in the E. coli HMS174(DE3) host strain was deposited in accordance with the Budapest Treaty with the 30 American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA on 8 October 1993 and assigned the accession number ATCC 69462. 61 B. Determination of genetic characteristics of the staphylococcal nuclease gene cloned in PSNUC24-26 In order to determine the genetic characteristics of the nuclease gene in pSNUC24-26, nucleotide sequence analysis was 5 performed by following the Sanger dideoxy chain termination method using the T7 promoter primer (SEQ ID NO:3) (5'-TAATACGACTCACTATA-3') and the T7 terminator primer 3'-TGGCGACTCGTTATTGATC-5' (this primer being complementary to the sequence 5'- ACCGCTGAGCAATAACTAG-3' (SEQ ID NO:4) ) . The 10 complete nucleotide sequence of the nuclease gene in pSNUC24-26 is presented in Fig. 3. A number of point mutations and addition of several base pairs were detected in the cloned staphylococcal nuclease gene in pSNUC24-26 resulting in a mutant form of the gene. A small DNA sequence, 5' -GGATCG-3' , 15 was inserted by an unknown mechanism immediately upstream of the BamHI site of the left side primer sequence during the PCR DNA amplification. Also, deletion of a nucleotide base, C, in the second position of the second codon [CCT > CT, i.e. C (AC) T] , and transition of A > G at the 4th and 36th codons (ACT > GCT at the 4th and AAG > GAG at the 36th positions) were evidenced.

Although, the deletion of a nucleotide base, C, at the second codon resulted in a frameshift mutation, the wild type amino acid residues of the nuclease protein were restored at the 25 3rd codon (GCG - Ala) and from the 5th codon (TTA - Leu) onwards. As a result, for the conformational stability and biological activities, the amino acid residues at positions 19 (GAT - Asp) , 21 (GAT - Asp) , 40 (GAC - Asp) , 41 (ACA -Thr) and 43 (GAA - Glu) that have been shown to be essential 30 for binding to Ca2+ (Tucker et al., 1979, Molecular and Cellular Biochemistry, 22, 67-86) together with the amino acid residues at positions 35 (AGA - Arg), 84 {AAA - Lys), 85 (TAT - Tyr) , 87 (CGT - Arg) and 113 (TAT - Tyr) that have been shown to be essential for intramolecular interactions to 35 establish the "active site" of the protein (Tucker et al., 1979), were not affected by this mutation (cf. Fig. 3.

WO 95/10614 PCT/DK94/00381 62 However, the changes in amino acid residues from Glu > Arg at position 10, from Pro > Thr at position 11 due to a single base deletion (CCT > CT) and the insertion of the DNA sequence 5'-GGATCG-3' upstream of the primer location, and 5 changes in the amino acid residues of Thr > Ala at position 13 and of Lys > Arg at position 36 due to the point mutations (A > G) of the wild type nuclease gene may have altered the extracellular transport and relative biological activities of the protein encoded by the plasmid pSNUC24-26.

C- Kinetics of the PSNUC24-26-encoded staphylococcal nuclease in E. coll HMS174(DE3) To determine the cell function-limiting (or killing) efficiencies of the mutant staphylococcal nuclease gene in the pSNUC24-26 plasmid, induction of the E. coli HMS174(DE3) 15 carrying this plasmid was performed by the addition of 1 mM of IPTG in an exponentially growing liquid culture. Before and after addition of IPTG (i.e. induction), aliquots of serially diluted culture were tested on DNAse Test Agar plates without IPTG. A decrease in the cell number at the 20 level of > 4 log was evidenced within the first 3 h time period following induction when plated onto DNAse Test Agar plates without IPTG (Fig. 4).

D. Intracellular stability of the mutant nuclease enzvme as determined bv immunological assay The stability of the mutant nuclease enzyme within the E. coli cells was determined by ELISA assay using polyclonal antibodies raised in rabbit against purified wild type Staphylococcus aureus (Foggi) nuclease. An exponentially growing culture of E. coli HMS174(DE3) carrying pSNUC24-26 was 30 induced by the addition of 1 mM of IPTG for 30 minutes.

Immediately after induction, 40 /*g per ml of chloramphenicol was added to the culture to inhibit protein synthesis and the cells were continued to grow for another 3 hours. Aliquots of the culture were separated at every 30 min interval for up to 63 PCT/DK94/003S1 3 h, centrifuged, washed 2 x with phosphate buffered saline (PBS) (pH 7.2), resuspended in PBS and the cells were disrupted by using a sonic disrupter to release the total cellular proteins. An aliquot of the proteins released at every 5 time period following chloramphenicol treatment was immobilized into a microtiter plate .and ELISA assay was performed as described in reference example 2.

About 2.5 fold increase in the nuclease protein was evidenced following induction and the nuclease protein was stable 0 within the cell > 1.5 h following chloramphenicol treatment (Fig. 5). Therefore, the intracellular stability of the mutant nuclease protein was not affected to the same extent as it was observed with the wild type nuclease protein. However, the basal level of production of nuclease was found 5 to be extremely low in the mutant staphylococcal nuclease protein suggesting that the modifications in the nucleotide sequences may have contributed to less extracellular transport of the mutant protein.

E. Identification of the mutant nuclease protein bv irmimno-0 logical assays For identification of the mutant nuclease protein in the pSNUC24-26 plasmid, western blot analysis was performed by using polyclonal antibody raised against purified wild type Staphylococcus aureus (Foggi) nuclease in rabbit. Total 5 cellular proteins in E. coli HMS174(DE3) harbouring pSNUC24-26 plasmid before and after IPTG induction were subjected to electrophoretic separation in denaturing polyacrylamide gel with appropriate size standards. The proteins were immobilized on a nitrocellulose membrane and subjected to western 0 blot analysis according to the procedure as described in reference example 1.

The western blot analysis r>f the mutant staphylococcal nuclease showed very weak aignal before induction. However, 64 following induction, the signal increased significantly. In the wild type clones, differences in the intensities of the nuclease enzyme before and after induction was indistinguishable. This suggests that the mutant nuclease gene encoded the 5 modified protein that may significantly have prevented the extracellular transport of the modified mutant nuclease.

F. Cloning of the mutant staphylococcal nuclease gene in the PS542Q pxagmid vector The intracellular effectiveness of the above mutant staphylo-10 coccal nuclease (snuc*) under the control of a different promoter element was tested by cloning the mutant staphylococcal gene with the upstream coding sequences under the control of the Ptac promoter/IacO (a synthetic promoter generated by the fusion of the tryptophan and the lactose 15 promoters) in pSE420 (Invitrogen Corp., 3985 B Sorrento Valley Blvd., San Diego, CA 92121). The Ncol and the Xhol restriction endonuclease-digested fragment from pSNUC24-26 plasmid consisting of the mutant staphylococcal nuclease gene with the upstream coding sequences was purified and ligated 20 to the similarly digested pSE420 plasmid vector and the resulting recombinant plasmid was designated pSNUC420-26. pSNUC420-26 was transformed into E. coli NM522 (Genotype = F' {proAB+ laclq lacZ AM15}, supE, thil, (lacproAB), hsd5 (r" m") X" (Invitrogen Corp.) pSNUC420-26 in the E. coli NM522 host strain was deposited in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA on 8 October 1993 and assigned the accession number ATCC 69463.

WO 95/10614 PCT/DK94/00381 65 G. Kinetics of mutant SNUC induction in E. coli NM522 (PSNUC420-26) The procedure for induction with IPTG and viable plate counts as described above for pSNUC24-26 was followed. The results 5 of the induction are presented in Fig. 6. A significant reduction of cell numbers (>5 log) within the first 1.5 h time period was evidenced.

H. Stability of the mutant nuclease in E. coli NM522 (PSNUC420-26) The intracellular stability of the mutant nuclease protein in pSE420-26 was determined by ELISA assay as described above for pSNUC24-26. The results are presented in Fig. 7. About 2-fold increase in the intracellular nuclease was evidenced following induction. Also, the intracellular stability of the mutant nuclease was determined to about 1.5 h following treatment with rifampicin. It was also observed that the basal level of nuclease expression during uninduced state was negligible. This result is comparable with that of the ELISA assay in pSNUC24-26.

I. Identification of the PSE420-26-encoded mutant nuclease bv western blot analysis Western blot analysis of the mutant staphylococcal nuclease in pSE420-26 showed low level of signal before induction and the higher signal following induction as observed above for 25 the pSNUC24-26 plasmid. 66 EXAMPLE 2 The use of the xth exonuclease III aene from Escheri -hia coli in combination with a mutant staphylococcal nuclease gene (snuc*) for the containment of genetically engineered micro-5 organisms The exonuclease III of E. coli has multiple catalytic activities in vivo: (i) it has 3'-5'exonuclease activities specific for the double stranded DNA, (ii) it can remove a number of 3'-end termini from duplex DNA, including 3'-phosphates, 10 (iii) it is an AP endonuclease which cleaves phosphodiester bonds at AP sites to yield base-free deoxyribose 5'-phosphate end groups, (iv) it has an RNase H activity which preferentially degrades the RNA strand in a RNA-DNA hybrid duplex (Weiss, 1981).

The exonuclease III of E. coli is encoded by the gene xth, the sequence of which has been determined and reported by Saparito et al. (1988).

The coding sequence including the ribosome binding site (SD sequence) of the xth gene was PCR amplified using two oligo-20 nucleotide primers L-XTH and R-XTH and cloned into the pCRII® vector and then cloned into the pSE420® plasmid vector (Nova-gen, Inc.) at the Xbal and Kpn restriction sites tinder the control of the trp-lac promoter (P^jJ/Iac operator {lacO) and 2aclq. The orientation of the cloned xth gene in pSE420 25 vector has been confirmed by restriction enzyme analysis. The Pt^/lacO; :xth gene from the pSE420 vector will be recovered by restriction enzyme digestion and cloned into pUCl8Not vector which already has the P^/lacOiisnuc* and the lacJ"9 gene.

Fallowing cloning, the 'BtBLC/lacOi ixth, P^^./lacO: i snuc* and lacl9 genes as a complete genetic cassette will be recovered from the pUC18Not vector as a Not! fragment and inserted into a similarly digested transposon vector pUTKM for insertion on WO 95/106X4 PCT/DK94/00381 67 the chromosome of the host microbial strain. In this system, both the staphylococcal nuclease gene (snuc*) and the exonuclease III (xth) gene can be expressed simply by addition of IPTG and the efficiency of killing of the host 5 strain can be determined by viable cell plate counts. During the normal physiological conditions (uninduced stage), both genes will remain silent due to the binding of the repressor protein encoded by the lacl gene to the lacO DNA sequence.

EXAMPLE 3 The construction of a replicon simultaneously expressing the snuc* and the hok genes The Ptag,/lacO:: snuc* and the lacl9 gene cassette was recovered from the pSNUC26-420 vector using the EcoRV and Xhol restriction enzymes and blunt ended by "fill in" reac-15 tion. This fraction was cloned in pBAP24ii vector (Bej et al., 1992) at the Sail restriction site which was also blunt ended by "fill in" reaction. The pBAP24h vector has the hok gene under the control of the P^/lacO promoter-operator and the lacJq. Upon induction with IPTG, both genes, snuc* and hok 20 can be expressed simultaneously. The cell function-limiting effects due to the simultaneous expression of the hok and the snuc* genes can be monitored by induction with ITPG and viable cell plate counts. The clones with the appropriate orientation of the genes was confirmed by restriction analy-25 sis. 68 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bis) A. Hie indications made below relate to the microorganism referred to in the description on page 60 „line 33 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet |X| Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (includingpostal code and country) 12301 Parklawn Drive Rockville MD 20852, USA Date of deposit 8 October 1993 Accession Number ATCC 69462 C. ADDITIONAL INDICATIONS (leave blank if net applicable) Tbis information is continued on an additional sheet □ As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications arc not for alt designated Siaia) E. SEPARATE FURNISHING o/INDICATIONS (UmeUaniifnot applicable) The indications listed belowwiil be submitted to tbe International Bureau later (specify the general naiurcofthein£ca6onsc.g^ 'Accession Number of Deposit") For receiving Office use only □ Tbis sheet was received with tbe international, application Authorized officer ■Pi a For International Bureau use only | | This sbeet was received by the International Bureau on: Authorized officer Form PCI7RO/I34 (July 1992> 69 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bis) A. Tbe indications made below relate to tbe microorganism referred to in tbe description on page _ 64 .line 29 .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet E Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (includingpostal code and country) 12301 Parklawn Drive Rockville MD 20852, USA Date of deposit 8 October 1993 Accession Number ATCC 69463 C. ADDITIONAL. INDICATIONS (leave blank if net applicable) This information is contb-Jed on an additional sheet □ As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications are notfor all designated States) E. SEPARATE FURNISHIN G OF^INDICATIONS (leave blank if net applicable Tbe indications listed below will be submitted to tbe International Bureau later (specify the general nature oftheindications eg^ 'Accession Number of Deposit0) For receiving Office use only 0 Tbis sheet was received with tbe international application Authorized officer For International Bureau use only □ Tbis sheet was received by tbe International Bureau ore Authorized officer Form PCT/RO/134 (July I992> 70 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Genexpress ApS (B) STREET: Motiisvej 70 (C) CITY: Holte ' (D) COUNTRY: Denmark (E) POSTAL CODE (ZIP): 2840 (ii) TITLE OF INVENTION: Recombinant Staphylococcal Nucleases and Their Use in Limiting the Survival □£ Genetically Engineering Microorganisms (iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GATCCGGATC CGCAACTTCA ACTAAAAAAT TACATAA 37 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGTACCGGAA TTCGTGCCAC TAGCAGCAGT GAC 33 71 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHAW*. -tfttSTICS: (A) LENGTH: 17 aase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) ~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TAATACGACT CACTATA 17 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACCGCTGAGC AATAACTAG 19 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 763 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 215..727 (D) OTHER INFORMATION: /notes "The initiation codon GTG, while normally translated as Valine, may also be translated as N-fonnyl Methionine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CGTAACCGGC TAGTTGCGGC CGCTGCCAGC CATTTGCCAC TCTCCTTTTC ATCCGCATCG 60 GCAGGGTCAT CCGGGCGCAT CCACCACTCC TGATGCAGTA ATCCTACGGT GCGGAATGTG 120 GTGGCCTCGA AATTCTGTCA TAAAGTTGTC ACGGCCGAGA CTTATAGTCG CTTTGTTTTT 180 72 ATTTTTTAAT GTATTTGTAC ATGGAGAAAA TAAA GTG AAA CAA ACG ACT ATT 232 Met: Lys Gin Thr Thr lie 1 5 GCA CTG GCA CTC TTA CCG TTA CTG TTT ACC CCT GTG ACA AAA GCC GCA 280 Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala Ala 10 15 20 ACT TCA ACT AAA AAA TTA CAT AAA GAA CCT'GCG ACT TTA ATT AAA GCG 328 Thr Ser Thr Lys Lys Leu His Lys Glu Pro Ala Thr Leu lie Lys Ala 25 30 35 ATT GAT GGT GAT ACG GTT AAA TTA ATG TAC AAA GGT CAA CCA ATG ACA 376 lie Asp Gly Asp Thr Val Lys Leu Met Tyr Lys Gly Gin Pro Met Thr 40 45 50 TTC AGA CTA TTA TTG GTC GAC ACA CCT GAA ACA AAG CAT CCT AAA AAA 424 Phe Arg Leu Leu Leu Val Asp Thr Pro Glu Thr Lys His Pro Lys Lys 55 60 65 70 GGT GTA GAG AAA TAT GGT CCT GAA GCA AGT GCA TTT ACC AAA AAA ATG 472 Gly Val Glu Lys Tyr Gly Pro Glu Ala Ser Ala Phe Thr Lys Lys Met 75 80 85 GTA GAA AAT GCA AAG AAA ATT GAA GTC GAA TTC GAC AAA GGT CAA AGA 520 Val Glu Asn Ala Lys Lys lie Glu Val Glu Phe Asp Lys Gly Gin Arg 90 95 100 ACT GAT AAA TAT GGA CGT GGG CTA GCG TAT ATT TAT GCT GAT GGA AAA 568 Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr lie Tyr Ala Asp Gly Lys 105 110 115 ATG GTA AAC GAA GCT TTA GTT CGT CAA GGC TTG GCT AAA GTT GCT TAT 616 Met Val Asn Glu Ala Leu Val Arg Gin Gly Leu Ala Lys Val Ala Tyr 120 125 130 GTT TAC AAA CCT AAC AAT ACA CAT GAA CAA CAT TTA AGA AAA AGT GAA 664 Val Tyr Lys Pro Asn Asn Thr His Glu Gin His Leu Arg Lys Ser Glu 135 140 145 150 GCA CAA GCG AAA AAA GAG AAA TTA AAT ATT TGG AGC GAA GAC AAC GCT 712 Ala Gin Ala Lys Lys Glu Lys Leu Asn lie Trp Ser Glu Asp Asn Ala 155 160 165 GAT TCA GGT CAA TAATGCTCAT TGTAAAAGTG TCACTGCTGC TAGTGGCAC 763 ABp Ser Gly Gin 170 (2) INFORMATION FOR SEQ ID NO:6 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 170 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 73 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Gin Thr Thr lie Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 15 10 15 Pro Val Thr Lys Ala Ala Thr Ser Thr Lys Lys Leu His Lys Glu Pro 20 25 30 Ala Thr Leu lie Lys Ala lie Asp Gly Asp Thr Val Lys Leu Met Tyr 35 40 45 Lys Gly Gin Pro Met Thr Phe Arg Leu Leu Leu Val Asp Thr Pro Glu 50 55 60 Thr Lys His Pro Lys Lys Gly Val Glu Lys Tyr Gly Pro Glu Ala Ser 65 70 75 80 Ala Phe Thr Lys Lye Met Val Glu Asn Ala Lys Lys lie Glu Val Glu 85 90 95 Phe Asp Lys Gly Gin Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr 100 105 110 lie Tyr Ala Asp Gly Lys Met Val Asn Glu Ala Leu Val Arg Gin Gly 115 120 125 Leu Ala Lys Val Ala Tyr Val Tyr Lys Pro Asn Asn Thr His Glu Gin 130 135 140 His Leu Arg Lys Ser Glu Ala Gin Ala Lys Lys Glu Lys Leu Asn lie 145 150 155 160 Trp Ser Glu Asp Asn Ala Asp Ser Gly Gin 165 170 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 482 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGGATCG GGATCCGAAC TGCGGCTTTA 60 ATTAAAGCGA TTGATGGTGA TACGGTTAAA TTAATGTACA AAGGTCAACC AATGACATTC 120 AGACTATTAT TGGTCGACAC ACCTGAAACA AGGCATCCTA AAAAAGGTGT AGAGAAATAT 180 GGTCCTGAAG CAAGTGCATT TACCAAAAAA ATGGTAGAAA ATGCAAAGAA AATTGAAGTC 240 GAATTCGACA AAGGTCAAAG AACTGATAAA TATGGAGCTG GGCTAGCGTA TATTTATGCT 300 GATGGAAAAA TGGTAAACGA AGCTTTAGTT TACAAACCTA ACAATACACA TGAACAACAT GAGAAATTAA ATATTTGGAG CGAAGACAAC CT 74 CGTCAAGGCT TGGCTAAAGT TGCTTATGTT 360 TTAAGAAAAA GTGAAGCACA AGCGAAAAAA 420 GCTGATTCAG GTCAATAATG CGAGCTCGAG 480 482 New Zealand Patent Application No. 274482 GX Biosystems A/S Mutant Nuclease Our ref: 13930 NZ 1

Claims (106)

5 CLAIMS

1. A cell containing a regulatably expressible gene coding for a truncated and/or mutated Staphylococcus aureus nuclease capable of intracellularly hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal peptide, 10 whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell membrane, the cell further containing a regulatory nucleotide sequence which regulates the expression of the gene.

2. A cell according to claim 1 wherein the expression of said 15 gene leads to formation of the nuclease in the cell at a rate which results in the presence of nicks in one strand of the cell nucleic acids to an extent which cannot be repaired by the nucleic acid repair mechanism of the cell, thereby limiting the function of said cell. 20

3. A cell according to claim l wherein the gene coding for the nuclease is contained in a recombinant replicon or recombinant replicons.

4. A cell according to claim 3 wherein the regulatory sequence is contained in a recombinant replicon or recoin- 25 binant replicons.

5. A cell according to claim 3 wherein the gene and the regulatory sequence are contained in the same recombinant replicon.

6. A cell according to claim 5 which contains a multiplicity 30 of replicons each containing both the gene and the regulatory sequence. 13390NZ1.C03/HS/199704 09 76 27 A 4$ 2

7. A cell according to claim 1 wherein the gene coding for a truncated and/or mutated Staphylococcus aureus nuclease is derived from Staphylococcus aureus (Foggi) ATCC 27355.

8. A cell according to claim 7 wherein the expression of the 5 gene results in the formation of a truncated nuclease.

9. A cell according to claim 8 wherein the truncated nuclease lacks at least 9 amino acid residues of those of the parent enzyme.

10. A cell according to claim 7 wherein the nuclease is 10 encoded by the DNA sequence of Fig. 3 (SEQ ID NO:7).

11. A cell according to claim 1 wherein the regulatory nucleotide sequence contains a regulatable promoter operably linked to the gene encoding the nuclease. 15

12. A cell according to claim 11 wherein the regulatable promoter is regulated by a factor selected from the environmental conditions of the cell, the physiological state of the cell and a stochastic event.

13. A cell according to claim 12 wherein the regulatable 20 promoter is regulated by a factor selected from the physical conditions in the environment and the presence or absence of a certain chemical in the environment.

14. A cell according to claim 13 wherein the chemical is selected from a carbon source, a nitrogen source, a metabo- 25 lite, an amino acid, a nucleoside, a pyrimidine base, a purine base, a metal ion and isopropyl-beta-D-thiogalactopy-ranoside (IPTG).

15. A cell according to claim 1 wherein the expression of the gene coding for a nuclease is stochastically induced as a 30 result of a recombinational excision of an excisable negatively functioning regulatory nucleotide sequence being 13390NZ1.C03/HS/199704 09 77 27 4 2 operably linked to said gene which nucleotide sequence, while present in the cell, inhibits expression of the gene whose expression results in the formation of the hydrolytically active enzyme. 5

16. A cell according to claim 15 wherein said excisable negatively functioning regulatory nucleotide sequence is a sequence which is flanked by a first flanking sequence and a second flanking sequence substantially homologous with said first flanking sequence whereby said regulatory element is 10 recombinationally excisable in the cell.

17. A cell according to claim 16 wherein the gene coding for a nuclease encodes a first RNA which is a messenger RNA, and the excisable negatively functioning regulatory nucleotide sequence being operably linked to said gene, is a gene 15 encoding a second RNA which forms an RNA-RNA duplex with said first messenger RNA and thereby, when it is expressed, inhibits translation of said gene coding for the hydrolytically active enzyme.

18. A cell according to claim 16 wherein the excisable nega-20 tively functioning regulatory nucleotide sequence is a gene encoding a polypeptide repressor of transcription of the gene coding for a nuclease.

19. A cell according to claim 18 wherein the excisable negatively functioning regulatory nucleotide sequence is a gene 25 encoding a lac repressor, and the gene coding for a nuclease is operably linked to the lac promoter, said lac promoter including the operator site for the lac repressor.

20. A cell according to claim 16 wherein the negatively functioning regulatory nucleotide sequence is a termination 30 sequence preventing transcription of the gene coding for a nuclease. i— • • ■ i ! - 6 A:;S mi 13390NZ1 .C03/HS/199704 09 ~T 78

21. A cell according to claim 1 wherein the expression of the gene coding for a nuclease is stochastically induced as a result of a site-specific recombinational excision of an excisable negatively functioning regulatory nucleotide 5 sequence being operably linked to said gene which nucleotide sequence, while present in the cell, inhibits expression of the gene coding for a nuclease.

22. A cell according to claim 21 wherein said excisable negatively functioning regulatory nucleotide sequence is a 10 sequence which is flanked by a first site for site-specific resolution and a second site for site-specific resolution, the second site being resolvable by the same or a functionally equivalent multimer resolving enzyme as is the first site,- whereby said regulatory element is recombinationally 15 excisable in the cell.

23. A cell according to claim 21 wherein the first and second site for site-specific resolution is the mrs site derived from plasmid RP4.

24. A cell according to claim 22 wherein the multimer resolv-20 ing enzyme is encoded by a gene which is located in trans relative to the sites for site-specific resolution.

25. A cell according to claim 24 wherein the gene coding for the multimer resolving enzyme is the parA gene of plasmid RP4. 25

26. A cell according to claim 21 wherein the gene coding for a nuclease encodes a first messenger RNA, and the excisable negatively functioning regulatory nucleotide sequence being operably linked to said gene, is a gene encoding a second messenger RNA which forms an RNA-RNA duplex with said first 30 messenger RNA and thereby, when it is expressed, inhibits translation of said gene. 13390NZI .C03/HS/199704 09 79 27 A 481

27. A cell according to claim 21 wherein the excisable negatively functioning regulatory nucleotide sequence is a gene encoding a polypeptide repressor of transcription of the gene coding for a nuclease. 5

28. A cell according to claim 27 wherein the excisable negatively functioning regulatory nucleotide sequence is a gene encoding a lac repressor, and the gene coding for a nuclease is operably linked to the lac promoter, said lac promoter including the operator site for the lac repressor. 10

29. A cell according to claim 21 wherein the negatively functioning regulatory nucleotide sequence is a termination sequence preventing transcription of the gene coding for a nuclease.

30. A cell according to claim 1 wherein the gene coding for a 15 nuclease is stochastically expressed as a result of recombinational inversion of an invertible promoter sequence of said regulatory nucleotide sequence said promoter being operably linked to the gene.

31. A cell according to claim 30 wherein the promoter 20 sequence is a sequence carrying the fimA promoter or a functional homologue thereof.

32. A cell according to claim 2 wherein at least one of (i) the gene coding for a nuclease and (ii) the nucleotide sequence regulating said gene is mutated at one or more 25 sites, whereby the cell function-limiting effect of the nuclease encoded by the gene, when expressed in the cell is the same or increased relative to the cell function-limiting effect of the enzyme expressed in a cell containing said gene and said nucleotide sequence in non-mutated form. 30

33. A cell according to claim 1 wherein the nucleotide sequence regulating the transcription of the gene coding for a nuclease is derived from a replicon selected from the group 13390NZ1.C03/HS/199704 09 27 A Aft 2 consisting of a bacterial plasmid, a bacterial chromosome, a prokaryotic virus, a eucaryotic plasmid, a eucaryotic virus, a eucaryotic chromosome, eucaryotic mitochondria, a eucaryotic chloroplast, and a synthetic sequence. 5

34. A cell according to claim l which comprises a further regulatably expressible gene which encodes a further gene product, said further gene product having a cell function limiting effect.

35. A cell according to claim 34 wherein the cell function-10 limiting gene product is an exonuclease including the gene product encoded by the E. coli xth gene. 3C.

A cell according to claim 34 wherein the further regulatably expressible gene is regulated by a regulatory nucleotide sequence of the same type as the sequence regulating 15 the gene coding for a nuclease.

37. A cell according to claim 34 wherein the further regulatably expressible gene is regulated by a regulatory nucleotide sequence of another type thar. the sequence regulating the gene coding for a nuclease, said other type being a 20 regulatory nucleotide sequence which is also capable of regulating the gene coding for a nuclease.

38. A cell according to claim 34 wherein the further regulatably expressible gene is selected from the hok gene from the parB region of plasmid R1 and a DNA sequence which is 25 functionally homologous to the R1 hok gene including the gef gene. 30

39. A cell according to claim 1 comprising a further DNA sequence not naturally related to the replicon carrying the gene coding for a nuclease and/or the replicon carrying the regulatory nucleotide sequence, said further DNA sequence being selected from a sequence coding for an active gene product, a sequence coding for a immunologically. -pesticidally ^ % -V , _ "U - v.;7 13390NZ1 .C03/HS/199704 09 *7 4*82 active gene product and a sequence coding for a pollutant-degrading gene product.

40. A cell according to claim 1 which is a cell transformed with a recombinant replicon or recombinant replicons con- 5 taining a gene coding for a nuclease, the expression of said gene leading to formation of the nuclease in the cell at a rate which results in hydrolysis of cell nucleic acids to an extent whereby the function of the cell is limited, the expression of said gene being regulated by a regulatory 10 nucleotide sequence which is contained in the recombinant replicon containing the gene or in another recombinant replicon present in the transformed cell.

41. A recombinant replicon comprising a regulatably expressible gene coding for a truncated and/or mutated 15 Staphylococcus aureus nuclease capable of intracellularly hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in a cell comprising the replicon, essentially cannot be translocated outside the cell membrane, 20 the expression of said gene being regulatable by a regulatory nucleotide sequence which is contained in the recombinant replicon or in another recombinant replicon present in a cell containing the replicon. 25

42. A recombinant replicon according to claim 41 which contains the regulatory nucleotide sequence.

43, A replicon according to claim 1 wherein the gene coding for a nuclease is derived from Staphylococcus aureus (Foggi) ATCC 27355. 30

44, A replicon according to claim 43 wherein the nuclease lacks at least 9 amino acid residues of those of the parent enzyme. 13390NZ1 .C03/HS/199704 09 82 27 A 48 2

45. A replicon according to claim 44 wherein the nuclease is encoded by the DNA sequence of Fig. 3 (SEQ ID NO:7).

46. A replicon according to claim 41 which is selected from pSNUC24-26 and pSNUC420-26. 5

47. A replicon according to claim 41 wherein the regulatory nucleotide sequence contains a regulatable promoter operably linked to the gene coding for a nuclease.

48. A replicon according to claim 47 wherein the regulatable promoter is regulated by a factor selected from the environ- 10 mental conditions of a cell containing the replicon.- the physiological state of the cell and a stochastic event.

49. A replicon according to claim 48 wherein the regulatable promoter is regulated by the presence or the absence of a chemical in the environment of a cell containing the repli- 15 con, said chemical being selected from a carbon source, a nitrogen source, a metabolite, an amino acid, a nucleoside, a pyrimidine base, a purine base, a metal ion and isopropyl-beta-D-thiogalactopyranoside (IPTG).

50. A replicon according to claim 41 wherein the expression 20 of the gene coding for a nuclease is stochastically induced as a result of a recombinational excision of an excisable negatively functioning regulatory nucleotide sequence which, while present in the cell, inhibits expression of the gene coding for the hydrolytically active enzyme. 25

51. A replicon according to claim 50 wherein said excisable negatively functioning regulatory nucleotide sequence is a sequence flanked by a first flanking sequence and a second flanking sequence substantially homologous with said first flanking sequence whereby said regulatory element is recom-30 binationally excisable in the cell. 13390NZ1 .C03/HS/199704 09 83 nt u 48 2 J7 4

52. A replicon according to claim 50 wherein the gene coding for a nuclease encodes a first RNA which is a messenger RNA, and the excisable negatively functioning regulatory nucleotide sequence is a gene encoding a second RNA which 5 forms an RNA-RNA duplex with said first mesnenger RNA and thereby inhibits translation thereof.

53. A replicpn according to claim 50 wherein the negatively functioning regulatory nucleotide sequence is a gene encoding a polypeptide repressor of transcription of the gene coding 10 for a nuclease.

54. A replicon according to claim 53 wherein the negatively functioning regulatory nucleotide sequence is a gene encoding the lac repressor, and the gene coding for a nuclease is operably linked to the lac promoter, said lac promoter 15 including the operator site for said lac repressor.

55. A replicon according to claim 50 wherein the negatively functioning regulatory nucleotide sequence is a termination sequence preventing transcription of the gene coding for a nuclease. 20

56. A replicon according to claim 41 wherein the expression of the gene coding for a nuclease is stochastically induced as a result of a site-specific recombinational excision of an excisable negatively functioning regulatory nucleotide sequence being operably linked to said gene which nucleotide 25 sequence, while present in the cell, inhibits expression of the gene.

57. A replicon according to claim 56 wherein said txcisable negatively functioning regulatory nucleotide sequence is a sequence which is flanked by a first site for site-specific 30 resolution and a second site for site-specific resolution, the second site being resolvable by the same or a functionally equivalent multimer resolving enzyme a s ,\is the first ~'' 1SS7 13390NZ1.C03/HS/199704 09 27 A A B 2 site, whereby said regulatory element is recombinationally excisable in the cell.

58. A replicon according to claim 56 wherein the first and second site for site-specific resolution is the mrs site 5 derived from plasmid RP4.

59. A replicon according to claim 57 wherein the multimer resolving enzyme is encoded by a gene which is located in trans relative to the sites for site-specific resolution.

60.. A replicon according to claim 59 wherein the gene coding 10 for the multimer resolving enzyme is the parA gene of plasmid RP4.

61. A replicon according to claim 56 wherein the gene coding for a nuclease encodes a first RNA which is a messenger RNA, and the excisable negatively functioning regulatory nucleo- 15 tide sequence being operably linked to said gene, is a gene encoding a second RNA which forms an RNA-RNA duplex with said first messenger RNA and thereby, when it is expressed, inhibits translation of said gene.

62. A replicon according to claim 56 wherein the excisable 20 negatively functioning regulatory nucleotide sequence is a gene encoding a polypeptide repressor of transcription of the gene coding for a nuclease.

63. A replicon according to claim 62 wherein the excisable negatively functioning regulatory nucleotide sequence is a 25 gene encoding a lac repressor, and the gene coding for a nuclease is operably linked to the lac promoter, said lac promoter including the operator site for the lac repressor. •

64. A replicon according to claim 56 wherein the negatively functioning regulatory nucleotide sequence is a termination 30 sequence preventing transcription of the gene coding for a nuclease. ' 13390NZ1 .C03/HS/199704 09 85 il A A 8 2

65. A replicon according to claim 41 wherein the gene coding for a nuclease is stochastically expressed as a result of recombinational inversion of an invertible promoter sequence of said regulatory nucleotide sequence said promoter being 5 operably linked to the gene.

66. A replicon according to claim 65 wherein the promoter sequence is a sequence carrying the fimA promoter cl a functional homologue thereof.

67.. A replicon according to' claim 41 wherein the gene coding 10 for a nuclease or the nucleotide sequence regulating said gene is mutated at one or more sites, whereby the cell function-limiting effect of the nuclease encoded by the gene, when expressed in the cell is the same or increased relative to the cell function-limiting effect of the nuclease 15 expressed in a cell not containing mutated DNA.

68. A replicon according to claim 67 wherein the gene product of the mutated gene is an enzyme having an enzymatic activity which is at least about 2-fold of that of the wildtype enzyme. 20

69. A replicon according to claim 41 which is a replicon comprising a further regulatably expressible gene encoding a cell function-limiting function.

70. A replicon according to claim 69 wherein the further regulatably expressible gene is regulated by a regulatory 25 nucleotide sequence of the same type as the sequence regulating the gene coding for a nuclease.

71. A replicon according to claim 69 wherein the further regulatably expressible gene is selected from the hok gene from the parB region of plasmid R1 and a DNA sequence which 30 is functionally homologous to the R1 hok gene including the gef gene. 13390NZI .C03/HS/199704 09 27 4 4 8 2

72. A replicon according to claim 69 wherein the cell function-limiting gene product is an exonuclease including the gene product encoded by the E. coli xth gene.

73. A replicon according to claim 41 which is a replicon 5 comprising a further DNA sequence not naturally related to the replicon carrying the gene coding for the nuclease and/or the replicon carrying the regulatory nucleo' ide sequence, said further DNA sequence being selected from a sequence coding for an immunologically active gene product, a sequence 10 coding for a pesticidally active gene product and a sequence coding for a pollutant-degrading gene product.

74. A population of cells consisting of a multiplicity of cells as defined in claim 1.

75. A cell population according to claim 74 comprising trans-15 formable cells having been transformed with the recombinant replicon as defined in claim 41, said replicon being capable of replicating in said cells.

76. A cell population according to claim 74 wherein the cells are bacterial cells. 20

77. A cell population according to claim 76 wherein the bacterial cells are selected from species whose natural habitat is a habitat selected from soil, surface water and plants.

78. A cell population according to claim 76 wherein the 25 bacterial cells are gram-negative bacterial cells.

79. A cell population according to claim 76 wherein the expression of the gene coding for a nuclease is regulatable by a repressor substance which can undergo a decay when said cells are released to a human or animal body or the outer 30 environment to an extent whereby the represser ,• substance is ." ' converted to a non-functional form, said repressor substance ! -S 1C07 I ' ' i Jv'l 13390NZ1 .C03/HS/199704 09 - ■""> 87 27 A 48 2 being present in the cells of the population in different amounts whereby as a result of said decay, the function of the cells of the population will be gradually limited.

80. A method of limiting the survival of a cell population in 5 a firr;L or a second environment which method comprises transforming the cells of said population with a recombinant replicon being replicated in the cells of the population and containing a gene coding for a truncated and/or mutated Staphylococcus aureus nuclease capable of intracellularly 10 nucleic acids, said gene not comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell membrane, the ceils further containing a regulatory nucleotide sequence being regulatable by a factor in said 15 first or second environment, the presence or absence of which regulates the expression of the gene, the expression of said gene leading to formation of the nuclease in the cells at a rate which results in the hydrolysis of cell nucleic acids to an extent whereby the function of the cells is being limited, 20 leading to a limitation of the survival of the cell population.

81. A method according to claim 80 wherein the cell population is the cell population as defined in claims 77-82.

82. A method according to claim 80 wherein the survival of 25 the cell population is limited in a first environment in which the gene is expressed, said cell population thereby being contained in said first environment.

83. A method according to claim 80 wherein the survival of the cell population is not limited when present in a first 30 environment, which first environment could change to a second environment physically and/or chemically distinct from the first environment, in which first environment the gene coding for a nuclease is not expressed, but the survival of which cell population is limited when transferred to a second 13390NZ1 .C03/HS/199704 09 88 274482 environment or when present in a physically and/or chemically changed first environment, where the gene is expressed.

84. A method according to claim 80 wherein the survival of a cell population is being limited by providing in the cells a 5 gene coding for a nuclease, which is operably linked to a regulatory nucleotide sequence encoding a repressor substance which can undergo a decay when said cells are released to a human or animal body or the outer environment to an extent whereby the repressor substance is converted to a non-10 functional form, said repressor substance being present in the cells of the population in different amounts whereby as a result of said decay, the function of the cells of the population will be gradually be limited.

85. A method of containing an extrachromosomal recombinant 15 replicon to a first kind of cell, where said replicon is naturally transferable to a second kind of cell, which method comprises providing on the recombinant extrachromosomal replicon a gene coding for a truncated and/or mutated Staphylococcus aureus nuclease a capable of intracellularly 20 hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal-peptide whereby the nuclease, when it is expressed in a cell, essentially cannot be translocated outside the cell meiiforane, the formation of said enzyme being at a rate which results in the hydrolysis of 25 cell nucleic acids to an extent whereby the function of the cell is being limited, said first kind of cells having or being modified to have a chromosomal replicon comprising a regulatory nucleotide sequence which inhibits the expression of said gene and thereby protects said first kind of cells, 30 said regulatory gene being lacking in said second kind of cell, whereby, if a cell of the second kind receives said extrachromosomal recombinant replicon said gene is expressed and has a function-limiting effect thereon. 13390NZ1 .C03/HS/199704 09 89 974482

86. A method according to claim 85 wherein the recomBinant replicon is a replicon as defined in claim 41 which does not contain a regulatory nucleotide sequence.

87. A method according to claim 85 wherein the expression of 5 the gene coding for a nuclease leads to formation of the nuclease in the cell at a rate which results in the presence of nicks in the cell nucleic acids to an extent which cannot be repaired by the nucleic acid repair mechanism of the cells, thereby limiting the function of said cell. 10 8a.

A method of limiting the survival of a cell population which comprises transforming the cells thereof with a recombinant replicon containing a regulatably expressible gene which, when expressed in a cell encodes a truncated and/or mutated Staphylococcus aureus nuclease capable of 15 intracellularly hydrolysing nucleic acids, said gene not comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell membrane, the formation of the enzyme in the cell being at a rate which 20 results in the hydrolysis of cell nucleic acids necessary for non-limited function of the cells, to an extent whereby the function of the cells is being limited, the expression of said genes or genes being induced as a result of recombinational excision of an excisable negatively func-25 tioning regulatory nucleotide sequence which, while present in the cells, inhibits expression of the gene coding for the nuclease, said negatively functioning regulatory nucleotide sequence being contained in the recombinant replicon or in an other recombinant replicon present in cells of the population 30 containing the replicon.

89. A method according to claim 88 wherein the recombinant replicon is the replicon as defined in claim 50. ;

90. A method of limiting tlje survival of a cell population, comprising transforming t£he cells thereof I : - j 13390NZ1 .C03/HS/199704 09 90 27 A 48 2 with a recombinant replicon containing a regulatably expressible gene which, when expressed in a cell encodes a truncated and/or mutated Staphylococcus aureus nuclease capable of intracellularly hydrolysing nucleic acids, said gene not 5 comprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell membrane, the formation of the nuclease in the cell being at a rate which results in the hydrolysis of cell nucleic acids to an extent 10 whereby the function of the cells is being limited, the expression of said gene being induced as a result of a site-specific recombinational excision of an excisable negatively functioning regulatory nucleotide sequence which, while present in the cells, inhibits expression of the gene coding 15 for the nuclease, said negatively functioning regulatory nucleotide sequence being contained in the recombinant replicon or in an other recombinant replicon present in cells of the population containing the replicon.

91. A method according to claim go wherein the recombinant 20 replicon is the replicon as defined in claim 69.

92. A method of limiting the survival of a cell population which comprises transforming the cells thereof with a recombinant replicon containing a regulatably expressible gene which, when expressed in the cells of the population 25 encodes a truncated and/or mutated Staphylococcus aureus nuclease capable of intracellularly hydrolysing nucleic acids, said gene not coitprising a sequence coding for a signal peptide, whereby the nuclease, when it is expressed in the cell, essentially cannot be translocated outside the cell 30 membrane, the formation of the nuclease in the cells being at a rate which results in the hydrolysis of cell nucleic acids necessary for non-limited function of the cells, to an extent whereby the function of the cells is being limited, the expression of said gene being expressed as arresult of 35 recombinational inversion of an invertible prolmoter sequence of said regulatory nucleotide sequence, saidjpromoter being _ /> /■•--V s-r.-y 13390NZ1 .C03/HS/199704 09 91 27 A Aft 2 operably linked to the gene coding for the enzyme which, while present in the cells, inhibits expression of the gene coding for the enzyme, said regulatory nucleotide sequence being contained in the recombinant replicon or in an other 5 recombinant replicon present in a cells containing the replicon.

93, A method according to claim 92 wherein the promoter sequence is a sequence carrying the fimA promoter or functional homologue thereof. 10

94. An immunologically active composition which contains a viable function-limited cell population as defined in claim 74 wherein the cells contain a further DNA sequence not naturally related to the gene coding for the nuclease or to the regulatory nucleotide sequence, which further sequence is 15 a sequence coding for an immunologically active gene product, the cells being function-limited to an extent which, when the composition is administered to a human or an animal, allows the cells to express the immunologically active gene product for a period of time and in an amount sufficient to obtain an 20 effective immune response in said human or animal, but which does not allow the cells to persist in the human or the animal. .

95# A composition according to claim 94 wherein the cells therein contain a sequence coding for an immunologically 25 active gene product which is a sequence coding for a fusion protein comprising the immunologically active gene product and a polypeptide, the presence of which results in the transportation of said fusion protein to the outer surface of the cells. 30

96. A composition according to claim 98 wherein the polypeptide being present in the fusion protein is a cell surface polypeptide selected from a polypeptide derived from fim-brillin protein, a pilus, a flagellum, an OM surface protein. 13390NZ1 .C03/HS/199704 09 92 27 A 48 2

97. A composition according to claim 95 wherein the cell surface polypeptide is derived from a bacterial species selected from Eaterobacteriaceae, Vibrionaceae and Pseudo-monadaceae. 5

98. A pesticidally active composition which contains a viable cell population as defined in claim 74 wherein the cells contain a further DNA sequence not naturally related to the gene coding for the hydrolytically active enzyme or to the regulatory nucleotide sequence, which further sequence is a 10 sequence coding for a pesticidally active gene product, the cells being function-limited to an extent which, when the composition is administered to an environment containing a pest, allows the cells to express the pesticidally active gene product for a period of time and in an amount sufficient 15 to obtain an effective pesticidal effect in said environment but which does not allow the cells to persist in the environment . ^

99. A composition according to claim 98 wherein the further DNA sequence.codes for a gene product which is toxic for 20 insects or their progeny.

100. A composition according to claim 99 wherein the further DNA sequence is a sequence derived from a strain of Bacillus thuringiensis encoding an insecticidal protein.

101. An environmental pollutant-degrading composition which 25 contains a viable cell population as defined in claim 74 wherein the cells contains a further DNA sequence not naturally related to the gene coding for the hydrolytically active enzyme or to the regulatory nucleotide sequence, which further sequence is a sequence coding for an environmental 30 pollutant-degrading gene product, the cells being function-limited to an extent which, when the composition is administered to an environment containing a pollutant -to be— - > degraded, allows the cells to express said poilutaiit degra- i ding gene product for a period of time and :.n an ajnpu.ht^ 13390NZ1 .C03/HS/199704 09 93 27 4 48 2 sufficient to obtain an effective pollutant-degrading effect in said environment but which does not allow the cells to persist in the environment.

102. A composition according to claim loi wherein the gene or genes the expression of which results in the formation of a cell function-limiting enzyme is only expressed when the pollutant degradable by the pollutant-degrading gene product is substantially degraded.

103. A cell containing a regulatably expressible gene coding for a truncated and/ or mutated Staphylococcus aureus nuclease substantially as herein described with reference to the accompanying Example.

104. A recombinant replicon comprising a regulatably expressible gene coding for a truncated and/or mutated Staphylococcus aureus nuclease substantially as herein described with reference to the accompanying Example.

105. A method of limiting the survival of a cell population as claimed in any one of claims 80, 88 or 92 substantially as herein described.

106. A method of containing an extrachromosomal recombinant replicon to a first kind of cell as claimed in claim 85 and substantially as herein described. 2ND OF CLAIMS ASPEC93128 13390NZ1.C03/HS/I99704 09