IE83441B1 - Process for the genetic manipulation of myxobacteria - Google Patents

Description

PATENTS ACT l992 920654 PROCESS FOR THE GENETIC MANIPULATION OF MYXOBACTERIA NOVARTIS AG The present invention relates to a novel process for the genetic manipulation of myxobacteria of the Sorangium/Polyangium group, which makes it possible for the first time specifically to apply recombinant DNA techniques to this group of organisms. invention relates to a In particular, present process for insertion of sequences of homologous or heterologous origin or a combination of DNA sequences of homologous or heterologous origin into the chromosome of said myxobacteria via homologous recombination, and to genetically modified myxobacteria prepared with the aid of this process.

DNA plasmids and vectors which are particularly suited for the the Likewise embraced are recombinant molecules, use in process according to invention, genetically modified myxobacteria of Sorangium/Polyangium group containing exogenous DNA of homologous and/or heterologous origin.

The myxobacteria of the Sorangium/Polyangium group are highly specialised organisms which are commonly detectable in soil samples, dead plant material or in animal dung. Characteristic of this group of microorganisms is their ability to utilise cellulose or cellulose—containing degradation products as sole C source. Another characteristic feature of this group is their ability to highly metabolites. produce active secondary A large number of strains from this group which, for example, are able to synthesise plant—microbicidal compounds have now been described. Particularly important in this connection are the so—called soraphens, macrocyclic compounds which have a beneficial biocidal spectrum against phytopathogenic microorganisms, but especially against phytopathogenic fungi. These compounds have very advantageous curative, systemic and ix) particular, preventive properties and can be employed to protect numerous crop plants [EP 0 358 606].

It is also known of other representatives of the group of myxobacteria that they are able to synthesise highly active compounds with antibiotic potency [Reichenbach et al (l988)]. Because of the importance of these compounds, there is a great interest in understanding the genetic bases of their synthesis in order thus to provide the possibility of being able specifically to influence these where appropriate.

The precondition for this is the provision of a process which makes possible direct, and preferably targeted, manipulation of these organisms using recombinant DNA techniques, for example by the targeted incorporation DNA into the genome of of novel genes or gene fragments or other sequences, including whole plasmids, the myxobacteria.

EP 0 372 230 describes processes for conjugative transfer of mobilisable vectors from E.coli into Gram—positive bacteria, in particular coryne bacteria.

A few representatives of the group of myxobacteria have also already been the subject of investigations in this direction. Special interest in this connection was primarily directed at .MyXococcus xanthus, which is a myxobacterium which has now been extensively researched and for which various gene transfer processes have already been described. Thus, for example, the coli phage P1 has been used very intensively, initially for the insertion of transposon Tn5 into the Myxococcus xanthus chromosome [Kaiser (1984); Kuner and Kaiser (l98l)] and then later for the transfer of genes cloned in Myxococcus xanthus back into the original E.coli host [O’Conner and Zusman (1983); Shimkets et al (1983)). Another example relates to the genetic manipulation of Myxococcus (l988)]. xanthus by transduction [Shimkets and Asher, Another process for gene transfer is based on the use of the plasmid RP4 which has a very wide host range. (1985) transferred via Breton et al were able to show that this plasmid can be conjugation from E.coli into Myxococcus xanthus, and there is stably integrated into the chromosome. Based on these properties, Breton et al (1986) (1987) and Breton and Guespin-Michel were able to integrate foreign genes into the chromosome of Myxococcus xanthus. Investigations by Jaoua et al (1987; 1989) revealed that the observed integration is based, with a high degree of probability, on a so— called site specific recombination. The latter is confined to particular sites, which have a narrow spatial restriction, within the Myxococcus Xanthus chromosome and is mediated by one or more so—called hot spots on the RP4 plasmid. In addition, it has emerged during the investigations carried out within the scope of the present invention that the previously known Myxococcus system discovered here cannot be applied to bacteria of the Sorangium/Polyangium group. It is assumed that these organisms lack the specific structural elements which are necessary for site specific recombination on their chromosomes. In addition, it has been found that no stable transposition takes place with these organisms either, for example on use of transposon Tn5. the the the the the The object which it was intended to achieve within scope of this invention thus related primarily to provision of a universally applicable process for genetic manipulation of myxobacteria of Sorangium/Polyangium group, which is free of abovementioned restrictions of the known processes and thus permits undirected or else, preferably, targeted insertion of genetic material into myxobacteria, independent of structural elements present on the myxobacterial chromosome or of specific transposition GVGITCS .

This can be achieved according to the invention, for example, by preparing genetic constructs, especially plasmid vectors, which by reason of their specific structure, can be inserted at the desired sites within the chromosome and which are not linked, as is the case in the previously known processes, to the organisation of the myxobacterium chromosome or of the (hot particular integration sites predetermined by functional plasmid used spots), or else dependent on transpositions of integrated transposons.

The present invention thus relates primarily to a process for the genetic manipulation of myxobacteria of the Sorangium/ Polyangium group, which is characterised in that genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin is inserted into the cell recombination at myxobacterial and integrated, via homologous random or else, when there is appropriate knowledge of the structural and functional organisation of the bacterial genome, specifically at a site, which is accurately defined on the basis of the homology present between the inserted DNA and DNA intrinsic to the bacteria, into the chromosome of said myxobacteria, independent of structural elements present on the myxobacterial chromosome or of specific transposition events.

The process according to the invention for the genetic manipulation of myxobacteria of the Sorangium/ Polyangium group is particularly characterised in that (a) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin, which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (b) genetic material which naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on myxobacterial chromosome and which is therefore artificially linked with the aid of rDNA techniques known per se to such homologous or DNA genetic material to be else essentially of which the flanked by said homologous or else essentially homologous DNA sections, the cell via homologous recombination, at a homologous sections, as a result inserted is is inserted into myxobacterial and there integrated, site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the bacteria, into the chromosome of said myxobacteria.

The integration of the genetic material into the bacterial chromosome via homologous recombination is mediated by one or more DNA sections within the DNA to be inserted, which are homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome. It is thus not linked to particular chromosomal structures and can in principle take place at any desired site within the bacterial chromosome.

The homologous DNA sections which can be used within the scope of the process according to the invention do identity with the corresponding sections on the myxobacterial chromosome able to the not necessarily have to have lOO% in order to be bring about required recombination event. On the contrary, it suffices for these DNA sections to be essentially homologous with the corresponding regions on the bacterial genome, that is to say when these have a and 100% between 90% and 100%. degree of homology of between 80% and Very particularly preferably Thus, “homologous” DNA sections are intended also to mean hereinafter those sections which do not have lOO% with the the myxobacterial chromosome but are at least “essentially identity corresponding regions on homologous” therewith.

It is possible in this connection for the homologous DNA to be organism itself or else sections isolated either from the target from related organisms, for example after fragmentation of the particular genome.

When the DNA sequence of the DNA sections on the myxobacterial chromosome which are intended in each case for integration is known, the corresponding DNA fragments which are homologous with or else at least essentially homologous with these chromosomal sections can, of course, also be prepared by synthesis.

The DNA sections of homologous origin which can be used within the scope of the present invention can moreover be either DNA sections of known sequence or else DNA obtainable at random, for after fragments example restriction digestion of homologous DNA.

When the structural and functional organisation of the appropriate parts of the bacterial genome is known, the process according to the invention thus makes possible for the first time a targeted, predictable modification of the genes present on the myxobacterial genome (in situ modification) by exchange of natural or artificially modified genes, gene fragments or other useful DNA sequences with homologous DNA sections within the bacterial genome. The process according to the invention furthermore makes possible a specific identification of the function of individual genes within the complete genome of myxobacteria of the Sorangium/Polyangium group by specific switching off of genes [gene disruption] or by complementation of genes which have previously been inactivated by application of other processes, especially of mutation processes.

Besides the targeted and thus very efficient the in situ modification of genes intrinsic to bacteria invention has a number of other possible applications (directed mutagenesis), the process according to such as, for example, the incorporation of additional gene copies in regions known to have a high expression rate, and elimination and thus switching off of unwanted genes. It is furthermore also possible now to think about the following further possible applications.

- Incorporation of strong or controllable promoters in front of genes, intrinsic to the bacteria, with interesting functions.

- Cloning of genes of various origin into myxobacteria of the Sorangium/Polyangium group.

— Cloning and expression of genes of various origin.

— Transfer of the inserted DNA back into another microorganisn1 such as, for example, into E.coli for further processing.

— Use of the heterologous DNA of an inserted vector firstly as radioactive probe for identifying the myxobacterium fragments adjacent to the site of integration, and secondly as pattern [template] for amplification within the [PCR]. scope of a. polymerase chain reaction The present invention further relates to a process for the preparation of genetically modified bacteria of the Sorangium/Polyangium group, which is characterised in that (al) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin, which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (a2) genetic material which sections naturally contains no which are homologous with or else at least essentially homologous with a corresponding region on the artificially linked with the and is therefore aid of such homologous or myxobacterial chromosome rDNA techniques se to DNA known per else essentially which the flanked by homologous or else essentially homologous DNA sections; the cell recombination, at a homologous sections, as a result of genetic material to be inserted is said is inserted into myxobacterial and there integrated, via homologous site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the the chromosome of said bacteria, into myxobacteria; and (b) positive transformants are selected with the aid of selection processes known per se and cultivated as pure culture.

The present invention thus makes possible for the first time a targeted genetic manipulation of the genome of myxobacteria of the Sorangium/Polyangium group, where the integration of the genetic material can be mediated, depending on the particular aim of the planned procedure, DNA alternatively either by homologous sections of known sequence or else by randomly selected homologous sections of unknown sequence.

The present invention likewise embraces recombinant DNA molecules which, by reason of their specific construction, are able to integrate genetic material such as, for example, genes or gene fragments or other useful DNA sequences which, where appropriate, code for novel and desirable properties, with the aid of homologous recombination at randoH1 or else, when the structural and functional organisation of the bacterial genome is known, also targeted at sites, which are accurately defined by reason of the homologies present between inserted DNA and DNA intrinsic to the bacteria, within the bacterial genome, as well as processes for the preparation of said recombinant DNA molecules.

The present invention furthermore embraces genetically modified myxobacteria of the Sorangium/Polyangium group with, where appropriate, novel and/or improved properties, which have been prepared by insertion of said recombinant DNA molecules.

The present invention additionally relates to the offspring of said modified myxobacteria and to mutants and variants thereof which still contain said recombinant DNA molecule.

A number of terms which are customary in recombinant DNA technology and in bacterial genetics are used in the following description.

In order to ensure clear and uniform understanding of the description and of the claims, as well as of the scope intended to apply to said terms, the following definitions are stated: Gene(s) or DNA of heterologous origin: A DNA sequence which codes for a specific product or products or fulfils a biological function and which originates from a species other than that into which the said gene is inserted; said DNA sequence is also called foreign gene or foreign DNA, or exogenous DNA.

Gene(s) or DNA of homologous origin: A DNA sequence which codes for a specific product or products or fulfils a biological function and which originates from the same species into which the said gene is inserted.

This DNA is also called exogenous DNA.

DNA homology: Degree of agreement between two or more DNA sequences. _ 10 _ fulfils a prepared by a for a specific product or products or biological function and which is synthetic route.

Promoter: A control sequence of DNA expression which ensures the transcription of any desired homologous or heterologous DNA gene sequence in a host cell, as long as said gene sequence is linked in an operable manner to a promoter of this type and the latter is active in said host cell.

Termination DNA sequence: at the sequence end of a transcription unit which signals end of the transcription process.

Promoter which is able to the Overproducing promoter (OPP): host cell sequence(s) bring about in a expression of any functional gene linked in an operable manner to an extent (measured in the form of the RNA or of the polypeptide amount) which is distinctly higher than is naturally observed in host cells which are not transformed with said OPP. //5/ located downstream/upstrean1 of the non—trans1ated region: DNA sections which are coding region and which, although transcribed in mRNA, This are not translated into a polypeptide. region contains regulatory sequences (5')- such as, for example, the ribosome binding site DNA expression vector: for Cloning vehicle such as, example, which contains all the expression of an inserted DNA in a suitable host cell. a plasmid or a bacteriophage, signal sequences which are necessary for which DNA transfer vector: Transfer vehicle such as, example, a plasmid or a bacteriophage vector, _.ll.. makes it possible to insert genetic material into a suitable host cell.

Homologous recombination: Reciprocal exchange of DNA pieces between homologous DNA molecules.

Mutants, variants: Spontaneously or else artificially, by application of known process measures such as, UV treatment, for example, treatment with mutagenic agents which still has the features and properties, essential to the etc , produced derivative of a microorganism invention, of the initial strain which has acquired the latter by reason of the transformation with exogenous DNA.

It has now been possible for the first time within the scope of the present invention to provide a process which makes possible a preferably targeted genetic the in that it is now possible manipulation of genome of myxobacteria of Sorangium/Polyangium group, for genetic material to be integrated, and also to be expressed therein, at randonx or else, when there is appropriate knowledge of the structural and functional organisation of the bacterial genome, defined predetermined in specifically at which the independent of structural elements accurately positions, can be some cases, within bacterial genome, the present on myxobacterial chromosome or of specific transposition events.

The process according to the invention is, moreover, the that it is possible to incorporate exogenous genetic material with essentially based on recognition the aid of homologous recombination into the genome of myxobacteria, it being possible to use, besides natural, also artificially modified and/or synthetic genes or gene fragments or other DNA sequences including whole plasmids, as long as they have DNA sections or are flanked by DNA sections which have a homology, which is sufficient for recombination, with corresponding sections on the myxobacterial genome.

The homologous DNA sections which can be used within the of the this connection necessarily have to have 100% identity with scope present invention do not in sections on the to be able recombination event. On the the corresponding myxobacterial about the contrary, it chromosome in order to bring required sections are suffices if these with the essentially homologous corresponding regions on the bacterial genome, that is to say if these have a degree 100% particularly preferably between 90% and 100%. of homology of between 80% and and very The size of said homologous regions can vary, but ought to be at least 100 Bp. of homology comprise between 0.3 Kb and 4 Kb, between 1 Kb and 3 Kb, Regions which but preferably are preferred within the scope of this invention.

Recombinant DNA molecules which, by reason of their specific construction, make possible the specific incorporation of genes or gene fragments or other interesting DNA sequences, including whole cell with the plasmids, into the genome of a target aid of homologous recombination in the abovementioned manner form an essential component of the present invention. invention relates in DNA The present particular to recombinant molecules which make possible a targeted integration of genetic material such as, for example, genes, gene fragments or other DNA fragments at a defined site within the genome of myxobacteria of which are the Sorangium/Polyangium and group, characterised in that they contain the DNA which is to be integrated, and in that said DNA has homologies with DNA regions within the else is flanked by corresponding myxobacterial genome, or such homologous DNA _l3_. sequences, to an extent such that, on transformation of the myxobacterial cell containing the homologous DNA region, there is undirected or else, preferably, targeted integration of said DNA, which is to be integrated, at a site, which is exactly defined by reason of the homology present between the inserted DNA and the DNA the the myxobacterial genome via homologous recombination. intrinsic to bacteria, within Said recombinant DNA molecules can be prepared very straightforwardly in such a way that the DNA which is to be which has the and abovementioned integrated properties (a) is isolated from a suitable source; or (b) when said DNA which is to be integrated naturally contains no sections which are homologous with or else at least with a essentially homologous corresponding region on the myxobacterial this DNA is artificially linked with the chromosome, aid of rDNA techniques known per se to corresponding homologous or else DNA which the genetic material to be inserted is flanked by DNA essentially homologous sections, as a result of said homologous or else essentially homologous sections.

If the DNA which is to be integrated is an expressable DNA sequence it is advantageous for the latter to be linked in an operable manner to expression signals capable of functioning in the bacterial cell, and, where appropriate, to be flanked by DNA sections which have homologies with a particular region within the bacterial genome. These flanking, homologous DNA sections are present, preferably fused together‘ to a unit, as component of DNA molecules which are closed in the form of a ring.

The latter can be dispensed with if said expressable DNA itself sufficiently great sequence with already has homology corresponding DNA regions within _l4_ sequence for said homologous genomic DNA can take place bacterial genome so that direct exchange of this by means of homologous recombination.

Besides double—stranded DNA, it is also possible to the single—stranded DNA and partially single—stranded DNA. employ in the process according to invention Suitable for use in the the invention are both homologous and heterologous gene(s) or DNA DNA sequences complying with the definition made within the process according to sequences, and synthetic gene(s) or scope of the present invention.

The DNA which be constructed exclusively from genomic, cDNA or DNA. Another possibility comprises the of hybrid DNA both of cDNA and of genomic DNA and/or synthetic DNA. sequences are to be integrated can, moreover, from synthetic construction sequences consisting In this case, the cDNA can originate from the same gene or DNA section as the genomic DNA or else both the CDNA and the genomic DNA can originate from different genes or DNA sections. In each case, however, it is possible for both the genomic DNA and/or the cDNA, each on its own, to be prepared fronm the same or front different genes or DNA sections.

If the DNA sequence contains portions of more than one gene or DNA section, these can derive either from one and the same organism, from a plurality of organisms, which belong to various strains, or varieties of the same species or different species of the same genus, or else from organisms which belong to more than one genus thereof or to another taxonomic unit.

In order to ensure the expression of a structural gene in the bacterial cell, it is possible, where appropriate, for the coding gene sequences initially to _l5_ be linked in an operable manner to expression sequences able to function in the myxobacterial cell. present the the which and, expressable hybrid constructions of thus gene invention usually contain, besides structural gene(s), also expression signals include both promoter and terminator sequences preferably, further regulatory sequences of the 3’ and ’ non—translated regions.

Every promoter and every terminator which is able to bring about induction of expression of an expressable DNA sequence in the myxobacteria of Sorangium/ Polyangium group can be used as a component of the hybrid gene construction.

Examples of promoters suitable for use in the process according to the invention are — the light—inducible [EP 310 619]; — other Myxococcus xanthus promoters; promoter of Myxococcus xanthus — promoters of Actinomycetes, especially of Strepto- mycetes; — E.coli promoters such as, for example, the Tac(hybrid), PL or Trp promoter.

Suitable termination sequences which can be used within the scope of this invention are described, for example, by Rosenber and Court (1979) and by Gentz et al (1981).

The functional unit which has been formed in this way and consists of a gene and of expression signals active in the myxobacterial cells can subsequently, where appropriate, be flanked by one or more DNA sections which have homologies with corresponding DNA regions within the myxobacterial genome to an extent such that, on transformation of the myxobacterial cell containing said homologous region, there is random or else, preferably, specific integration of a gene sequence which is flanked by homologous DNA sequences at a site, which is defined on the basis of the homologies present between inserted DNA and DNA intrinsic to the bacteria, the bacterial The within within genome by homologous flanking, DNA the this preferably fused together to a recombination. homologous sections are, moreover, scope of invention present, unit, as a component of a DNA molecule which is closed in the form of a ring.

In a preferred embodiment, to be moreover, the DNA which is inserted is integrated into a plasmid which either already contains homologous DNA sections or else acquires the latter cloned in at a later time.

Thus, if the intention is to integrate not just single genes or gene fragments but the complete plasmid DNA, which may contain said genes or gene fragments, into the myxobacterial genome, it is sufficient to clone said homologous DNA sequences into the plasmid DNA at a required site, although, where possible, the genes intended for expression should be functionally retained. Thus, it is also possible in this case too for the DNA which is to be integrated (plasmid DNA) to be regarded in principle as flanked by homologous DNA sequences because these homologous DNA sections can be thought of as fused together to a unit within the DNA molecule which is closed in the form of a ring.

Besides structural genes, it is also possible to use any other desirable genes or gene fragments or other useful DNA sequences such as, for example, binding sites of regulator molecules, promoters, terminator sequences etc.

It is now possible for the first time, by the choice of suitable DNA sequences of homologous or heterologous origin, which have sufficiently great homologies with corresponding sections within the bacterial genome and thus allow exchange of genetic material via homologous recombination, for genes or other DNA sequences to be integrated specifically at predetermined sites in the myxobacterial genome and to be expressed there where appropriate.

The extent of the homology, which is necessary for exchange via homologous recombination, between the homologous DNA sections and the corresponding genomic DNA region depends on a variety of parameters and must therefore be adapted to the appropriate needs in each the DNA used. It is that a least 100 Bp is case, depending on sequences assumed on the present state of knowledge homologous region comprising at sufficient to bring about the required recombination event.

A homologous region which extends over a range of 0.3 to 4 Kb, but preferably over a range of 1 to 3 Kb, is preferred within the scope of this invention.

Suitable for use as homologous DNA sections within the scope of this invention are primarily DNA sequences of homologous origin which can be obtained by isolation of the complete myxobacterial DNA and subsequent digestion the DNA sequence of said homologous DNA fragments is known they with suitable restriction enzymes. Where can, of course, also be prepared by synthesis.

However, it is furthermore also possible to use homologous DNA sections of heterologous origin, which have been isolated not directly from the genome of the but, for related organisms and which thus do not necessarily have 100% identity with the target organism example, from corresponding DNA regions on the genome of the target organism but are only essentially homologous with the latter, that is to say have a degree of homology between 80% and 100%. and very particularly preferably between 90% and l00%.

An essential the therefore formed by a process for the specific genetic the characterised in component of present invention is manipulation of myxobacteria of Sorangium/ group; that genetic material of homologous origin or a combination Polyangium which is of genetic material of homologous and heterologous origin is inserted into the myxobacterial cell and integrated there via homologous recombination specifically at a site, which is accurately defined by reason of the homologies present, into the chromosome of said myxobacteria.

It is thus now possible for the first time within the scope of this invention, by preparing appropriate hybrid gene constructions in the manner described above, to carry out specific modifications of bacteria- intrinsic genes within the myxobacterial genome or else to incorporate additional genes or other DNA fragments into the myxobacterial genome. If the integration takes place within a functional gene or operon, this usually leads to inactivation thereof and, as a consequence, to a phenotypically observable defect.

The specific procedure for this can be such that the of the with the myxobacterial cells are transformed with one recombinant DNA molecules described above, genes, hybrid gene constructions or other DNA fragments contained in said recombinant DNA molecule being integrated by homologous recombination randomly or else, preferably, specifically at a site, which is defined by reason of the homologies present and thus can be predetermined, into the bacterial genome.

In a specific embodiment of the present invention, the insertion of the genetic material into myxobacteria of the Sorangium/Polyangium group, takes place in an undirected manner via a donor organism capable of conjugation—like information exchange with the _19_. myxobacterium. target organism from the donor to the myxobacterium recipient.

The procedure for the preparation of suitable plasmids which have a homology with the myxobacterial chromosome which is sufficient for integration via homologous recombination can be, for example, such that the complete DNA is initially isolated from. myxobacteria and subsequently fragmented. This fragmentation can be carried out either mechanically by the action of shear forces or else, preferably, by using suitable restriction enzymes.

It is then possible to isolate from the large number of resulting fragments those of suitable size and subsequently clone thent into a suitable plasmid. The ligation of homologous DNA fragments and of DNA fragments of homologous and heterologous origin into a suitable cloning vector is carried out with the aid of standard methods as for . are described, example, by Maniatis et al, This usually entails the vector and the DNA sequence which is to be integrated initially being cut with suitable restriction enzymes. Examples of suitable restriction enzymes are those which provide fragments with blunt ends, such as, for example, Smal, Hpal and EcoRV, or else enzymes which form cohesive ends, such as, for example, EcoRI, Sacl, BamHI, Sall, Pvul etc.

Both fragments with blunt ends and those with cohesive ends, which are complementary with one another, can be linked again, with the aid of suitable DNA ligases, to give a single continuous DNA molecule.

Blunt ends can also be prepared. by treatment of DNA fragments which have protruding cohesive ends with the Klenow fragment of E.coli DNA polymerase by filling in the gaps with the appropriate nucleotides. complementary On the other hand, cohesive ends can also be prepared artificially, for example by attaching complementary homopolymer tails to the ends of a required DNA sequence and of the cut vector molecule using a terminal deoxynucleotidyl transferase or else by attaching synthetic oligonucleotide sequences (linkers) which carry a restriction cleavage site, and subsequent cutting with the appropriate enzyme.

It is possible in principle to use for the preparation and multiplication. of the constructs which have been described above and which contain DNA fragments of homologous or else a combination of DNA fragments of all example, homologous and heterologous origin conventional cloning vectors such as, for plasmid or bacteriophage vectors as long as they have replication which which are compatible with the host cell. and control sequences originate from species replication, cloning vector usually carries an origin of in addition specific genes which lead to phenotypical selection features in the transformed host cell, transformed vectors especially to resistance against antibiotics. The can be selected on the basis of these phenotypical markers after a transformation into a host cell.

Selectable the markers which can be used this phenotypical within scope of invention comprise, for example, without this representing a limitation on the the tetracycline, subject—matter of invention, resistances to ampicillin, G418, chloramphenicol, hygromycin, kanamycin, neomycin and bleomycin. A prototrophy particular amino acids can function as further selectable marker, for example.

Preferred within the scope of the present invention are the plasmid pSUP202l used within the scope of the present primarily E.coli plasmids such as, for example, invention.

Suitable host cells for the cloning described above which are within the scope of this invention are primarily prokaryotes, including bacterial hosts such as, for example, A.tumefaciens, E.coli, S.typhimurium and Serratia marcescens, furthermore pseudomonads, actinomycetes, salmonellae and myxobacteria themselves.

E.coli hosts such as, the E.coli for example, strain HBlOl are particularly preferred.

Competent cells of the E.coli strain HBlOl are in this with the aid of the customarily used for the transformation of E.coli connection prepared processes [seez “General recombinant DNA techniques”].

Transformation and subsequent incubation on a suitable medium are followed by the resulting colonies being subjected to differential screening by plating out on selective media. It is then subsequently possible to isolate the appropriate plasmid DNA from those colonies which contain plasmids with DNA fragments cloned in.

Recombinant plasmids of different size are obtained in this After then possible for plasmids of suitable size to be selected for the way. restriction analysis it is subsequent insertion of the plasmid DNA into the myxobacterial cell. This DNA transfer can moreover take place either directly or else, preferably, via an intermediate host (donor cell) capable of conjugation- like information exchange with the myxobacterium target organism within the scope of a conjugal transfer. the invention An essential component of present therefore relates to construction of plasmids _22_ which, besides homologous sections, can also contain one or more gene constructions consisting’ of one or more structural genes or other desirable genes or gene linked signals able to function The homologous DNA fragments can in this connection either fragments which are, where appropriate, in an operable manner to expression in bacterial cells, or other useful DNA sequences. consist entirely of genome 4 which are intrinsic to the the and thus are completely bacteria (myxobacteria of Sorangium/Polyangium group) or else of homologous origin, they can, besides homologous also sections, contain more or less expressed portions of heterologous origin. The use of homologous DNA sections of purely heterologous origin is also conceivable.

These plasmids can be used in a further process step for insertion of the genetic constructions which have which contain, which into the myxobacterial cell and been described above and where appropriate, a structural gene codes for a required gene product, integration there into the bacterial genome.

The transfer of the genetic constructions according to the application into the myxobacterial cells can be carried out in a variety of ways. Preferred within the of this cell scope invention is conjugal transfer fron1 a donor capable of conjugation—like information exchange with the myxobacterium target organism to the myxobacterial recipient.

It is possible within the scope of this conjugal transfer for the DNA which is to be transferred moreover to be either initially cloned, as described above, in one of the cloning vectors customarily used, suitable and subsequently transformed into a donor cell. host intermediate host which functions as the avoided by using a host strain which is suitable both roundabout route via intermediate can be -23.. for the cloning of DNA and for the use as donor cell within the scope of the conjugation.

Intermediate hosts which can be used within the scope of this invention as donor cells are essentially prokaryotic cells selected from the group consisting of E.coli, pseudomonads, actinomycetes, salmonellae and myxobacteria themselves.

The precondition for conjugal transfer of plasmid DNA from a. donor cell to a. recipient is (tra) the least the transfer origin the presence of transfer and mobilisation functions (mob).

Moreover mobilisation function must contain at (oriT) and be located on the plasmid to be transferred. (tra) By contrast, the transfer function can be either located on the plasmid or on a helper plasmid or else be present integrated into the chromosome of the donor cell.

Plasmids which meet the abovementioned precondition and are therefore preferred within the scope of this invention essentially fall into incompatibility groups P, Q, T, N, W and Coll. The prototype of the P group plasmids is the plasmid RP4. the scope of this pSUP202I which plasmid RP4, which has as component of the mob function (RP4mob) the origin Other with the mob function (RP4mob), for example, pSUPlOl, pSUP30l, pSUP401, pSUP20I, pSUP202, pSUP203 or pSUP205, and the derivatives derived therefrom et al (l988)] the process according to the invention.

Particularly preferred the 1.9 Kb fragment from the within invention is plasmid contains a (oriT). transfer plasmids such as, [Simon can likewise be used within the scope of During the course of the experiments carried out within the scope of this invention it has emerged that it is the exposed to a brief heat treatment during the course of advantageous when myxobacterial recipient is the conjugal transfer‘ before the incubation with the donor strain. A preincubation of the recipient cell at a temperature of 35°C to 60°C, preferably at a temperature of 42°C to 55°C and very particularly preferably at a temperature of 48°C to 52°C for one to minutes, but in particular 5 to 20 minutes, is preferred.

Used in a preferred embodiment of the present invention is an E.coli donor strain which contains the transfer genes (tra) of plasmid RP4 incorporated into the chromosomal DNA. Preferred within the scope of this invention is the E.coli donor strain W3lOl(pME305) which contains the helper plasmid pME305 which has the transfer function (tra) of RP4.

Particularly interesting for the process technique, and thus particularly preferred within the scope of this invention, are bacterial strains which are suitable both as hosts for cloning of vectors with integrated DNA sequences and for use as donor cell within the scope of the conjugal transfer. Likewise particularly preferred are bacterial strains which are restriction- negative and thus do not degrade inserted Both of the E.coli strain ED8767(pUZ8) foreign DNA. met by the but this abovementioned criteria are in an ideal manner, is nentioned at this point only as representative of other suitable bacterial strains and is not intended to limit the application in any way.

Besides the conjugal gene transfer, described above, frmn a donor cell into a myxobacterial recipient, it is, of course, also possible to use other suitable gene transfer processes for inserting genetic material into myxobacteria of the Sorangium/Polyangium group. Mention may be made here primarily of gene transfer via electroporation, within the scope of which the myxobacterial cells are briefly exposed to high electric field strengths [Kuspa and Kaiser (l989)]. The general outline conditions for electroporation of _25_ prokaryotic cells described in detail in US- P 4,910,140.

NON-LIMITING EXEMPLARY EMBODIMENTS General recombinant DNA techniques Since many of the recombinant DNA techniques used in this invention are routine for the person skilled in the art, a brief description of these generally used techniques is to be given below. All these processes A the reference of Maniatis et al are described in (1982), unless separate reference is made thereto.

A. Cutting with restriction endonucleases about 50 to 500 ug/ml DNA in the buffer solution recommended by the Typically, the reaction. mixture contains manufacturer, primarily New England Biolabs, Beverly, (FRG). 2 to 5 units of restriction endonucleases are added for each pg of DNA the the temperature recommended by the manufacturer for one to MA. and Bohringer, Mannheim and reaction mixture is incubated at three hours. The reaction is stopped by heating at 65°C followed ethanol. This technique is also described on pages lO4 to 106 of the (1982) for lO minutes or by extraction with phenol, by precipitation of the DNA with Maniatis et al reference.

B. Treatment of the DNA with polymerase in order to generate blunt ends to 500 pg/ml DNA fragments are added to a reaction mixture in the buffer recommended by the manufacturer, primarily New England Biolabs, Beverly, MA. and Bohringer, Mannheim (FRG). The reaction mixture contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction is carried out at 15°C for 30 minutes and is then stopped by heating at 65°C for 10 obtained by minutes. For which with fragments are endonucleases EcoRI cutting restriction and of DNA polymerase is used. For fragments which are obtained by which generate 5’-protruding ends, such as BamHI, the large fragment, or Klenow fragment, endonucleases which generate 3’—protruding ends, such as PstI and Sacl, T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al (1982) reference.

C. Agarose gel electrophoresis and purification of DNA fragments from gels The agarose gel electrophoresis is carried out in a horizontal apparatus as described on pages 150 to 163 The buffer used is the The DNA ethidium. bromide gel or tank buffer added the The DNA is visualised by illumination of the Maniatis et al reference. buffer stained by 0.5 pg/ml tris—acetate described therein. fragments are either in the which is present during electrophoresis or after electrophoresis. with long-wavelength ultraviolet light.

When the fragments are to be removed from the gel, the agarose used is one which gels at low temperature and can be obtained from Sigma Chemical, St Louis, Missouri. After the electrophoresis, the required fragment is cut out, placed in a plastic tube, heated at 65°C for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This process is a slight modification of that described by Maniatis et al (1982) on page 170.

As alternative, the DNA can be isolated from the agarose with the aid of the Geneclean kit (Bio 101 Inc , La Jolla, CA, USA).

D. Addition of synthetic linker fragments onto DNA ends -27..

If it is required to attach a new endonuclease cleavage site onto the end of a DNA molecule, the molecule is, treated with DNA generate blunt ends as described About 0.1 to 1.0 ug of the about 10 ng of phosphorylated been obtained from New England where appropriate, initially polymerase in order to section. added to which has in the above fragment is linker DNA, Biolabs, in a volume of 20 to 30 pl with 2 pl of T4 DNA and l mM ATP in the buffer recommended by the manufacturer. ligase from New England Biolabs, After the stopped by heating at 65°C for l0 minutes. incubation at 15°C overnight, reaction is The reaction mixture is diluted to about lOO ul in a buffer which is correct for the restriction endonuclease which cuts the synthetic linker sequence. Approximately 50 to 200 units of this endonuclease are added. The ndxture is incubated at the appropriate temperature for 2 to 6 hours, and then the fragment is subjected to an agarose gel electrophoresis and purified as described above.

The resulting fragment will now have ends with endings which with the restriction have been generated by These cutting endonuclease. ends are usually cohesive so that the resulting fragment can now easily with the cohesive be linked to other fragments same ends.

E. Removal of 5’-terminal phosphates from DNA fragments the the During the plasmid cloning treatment of with steps, vector plasmid phosphatase reduces (discussed on page l3 After the DNA has recircularisation of the vector of the Maniatis et al reference). been cut with the correct restriction endonuclease, one unit of alkaline phosphatase from the intestine of calves, which has been obtained from Boehringer— Mannheim, Mannheim, is added. The DNA is incubated at °C for one hour and subsequently twice extracted with phenol and precipitated with ethanol. _28_ F. Linkage of the DNA fragments When fragments with complementary cohesive ends are to be linked together, about 100 ng of each fragment are incubated ixu a reaction nuxture of 20 to 40 ul with about 0.2 of T4 DNA ligase Biolabs in the buffer recommended by the manufacturer. units from New England The incubation is carried out at 15°C for 1 to 20 hours. When DNA fragments with blunt ends are to be linked, they are incubated as above apart from the amount of T4 DNA ligase being increased from 2 to 4 units.

G. Transformation of DNA into E.coli The E.coli strains HBlOl, W3lOl and ED8767 are used for most of the experiments. DNA is introduced into E.coli by the calcium chloride process as has been described by Maniatis et al (1982), pages 250 to 251.

H. Screening of E.coli for plasmids After the the colonies of of the transformation, tested for resulting E.coli the presence required plasmid by a rapid plasmid isolation process. Two usual 366 to 369 of the processes are described. on pages Maniatis et al (1982) reference.

I. Isolation of plasmid DNA on a large scale Processes for the isolation of plasmids from E.coli on a large scale are described on pages 88 to 94 of the (1982) Maniatis et al reference.

Examples: Example 1: Cultivation conditions for Sorangium in a G5lb liquid Sorangium cellulosum is brought up -29..

“Media The It is also possible to use a G52c section and °C. shaking at 180 rpm. medium [see buffers”] at a temperature of cultures are aerated by medium as alternative medium.

“Media cultivation on The SolE mediun1 described in the section and buffers” the medium. The incubation temperature is 30°C in this case can be used for solid too.

Example 2: Cultivation conditions for E.coli E.coli cells are cultivated in an LB medium [Miller (1972)] at a temperature of 37°C.

Example 3: Preparation of a streptomycin—resistant spontaneous mutant of Sorangium cellulosum pl of a three—day old Sorangium cellulosum culture which has been raised in (So1E pg/ml (wild—type strain So ce 26] plated which is The incubation °C. The spontaneous solid medium with 300 liquid medium is out on medium] supplemented streptomycin. time is 14 days at a temperature of colonies growing on this medium are streptomycin-resistant mutants which are cultivated once more on the same medium (with streptomycin) for further concentration and purification.

One of these streptomycin—resistant colonies is selected and is called SJ3. A sample of this mutated Sorangium cellulosum So ce 26 strain was deposited on .01.1991 at the Mikroorganismen und Zellkulturen GmbH” FRG], provisions “Deutsche Sammlung Von [Braunschweig, which is recognised in accordance with the of the under deposit number DSM 6380.

Budapest Treaty as international depository, Example 4: Preparation of the complete DNA of Sorangium To isolate the complete DNA, a Sorangium culture in the stationary phase is centrifuged at 10,000 rpm for -30.. minutes. The cells are removed and resuspended in STE buffer [see section “Media and buffers”] to a cell density of about 109 cells/ml. and adjusted Subsequently 450 pl of this suspension are mixed with 200 pl of RLM buffer [see section “Media and buffers”] and 2 pl of diethyl pyrocarbonate. Thorough and uniform mixing are ensured by using suitable equipment such as, After incubation in for example, a Vortex or the like. an incubator at 70°C for 30 minutes, 100 pl of potassium acetate [5 M] are added. This mixture is incubated on ice for 15 ndnutes and thoroughly mixed [Vortex] every 5 minutes. After centrifugation [15 minutes at 10,000 rpm] the supernatant is subsequently mixed with 500 pl of phenol/chloroform/isoamyl alcohol [25/24/1] for extraction of the proteins. After renewed centrifugation [15 minutes at 10,000 rpm] the upper phase which contains the DNA fraction is removed, and any phenol content still present therein is removed with diethyl ether [1 ml]. The DNA is then precipitated by adding 1 ml of ethanol. After incubation at -70°C for 30 minutes, the complete mixture is centrifuged at ,000 rpm for 15 nunutes, 70% ethanol and dried in vacuo. dissolved in TER buffer. the pellet is washed with Finally, the DNA is Example 5: Conjugative transfer of pSJB55 into Sorangiumcellulosum SJ3 .1 Two-stage process .1.1 Cloning of Sorangium DNA into plasmid pSUP202l The chromosome isolated from the Sorangium SJ3 strain is cut with the restriction enzyme Pvul. this into the [Simon R et al This entails 0.2 pg of The fragments obtainable in are cloned pSUP2021 (1983)). plasmid DNA and 1 pg of chromosomal DNA being initially way plasmid digested with Pvul and subsequently precipitated with ethanol. The precipitate is removed, dried and the dried pellet is suspended in 14 pl of double-distilled water. Then 2.5 pl of a ten—fold concentrated ligation buffer “Media and buffers”], 2.5 pl of bovine serum albumin [O.1%], 2.5 pl of ATP [10 mM], 2.5 pl of DTT [O.2 M], 8 pl of H20 and 1 pl of T4 DNA ligase added. The incubated at a temperature of about 8°C overnight. [see section are complete ligation mixture is then pl of this ligation mixture are transformed into the strain HBlO1 for the this, competent E.coli recombinant the strain HB10l are prepared with the aid of the processes cloning of plasmids. For cells of E.coli normally used for the transformation of E.coli (see: “General recombinant DNA techniques”].

After transformation and subsequent incubation for 24 [25 pg/ml] the resulting colonies hours on LB agar supplemented with kanamycin [25 Hg/ml], are subjected to a differential screening by parallel [60 pg/ml] It is subsequently possible to and chloramphenicol plating out on ampicillin-containing and ampicillin—free medium. isolate those colonies which have lost their ampicillin resistance due to the integration of the Sorangium DNA are then isolated. from these fragments. The plasmids ampicillin—sensitive colonies.

Recombinant plasmids of different size are obtained in this way. After restriction analysis, three of these plasmids are selected for further experiments. These plasmids, called pSJB50, pSJB55 and pSJB58, contain Sorangium DNA inserts of 1 Kb, 3.5 Kb and 4 Kb respectively. .1.2. Conjugative transfer of plasmid pSJB55 into Sorangium cellulosum The transfer of plasmid pSJB55 into Sorangium cellulosum takes of E.coli strain W3101(pME305) place with the mediation (1987)], capable of a conjugation-like information exchange with [Jaoua S et al which is _32_ Sorangium. The E.coli plasmid pME305 [Rella (l984)] is in this case used as helper plasmid for the mobilisation of pSJB55.

Initially, competent cells of the E.coli strain W3l0l(pME305) are transformed, with the aid of the processes normally used for the transformation of E.coli, with 5 ;H_ of the previously isolated pSJB55 plasmid DNA. The transformed E.coli cells thereby become the donor for the plasmid pSJB55.

For the actual transfer, 15 ml of a Sorangium cellulosum SJ3 culture (4 x lOB cells/ml to 1-4 x lO9 cells/ml] in the stationary phase are mixed with 10 ml of a late log phase culture of E.coli donor cells which contain a comparable content of cells. These are then centrifuged together at 4000 to 8000 rpm for 10 minutes and resuspended in 500 pl of a G5lb or G5lt medium.

It has proved advantageous for the Sorangium recipient cells to be exposed briefly to a heat treatment in a waterbath before the conjugation with E.coli. The best transfer results with the Sorangium cellulosum strain SJ3 can be achieved with a heat treatment at a temperature of 50°C for l0 minutes. Under these conditions transfer frequencies of 1-5 x l0% can be achieved, which corresponds to an increase by a factor of 10 compared with. a process without previous heat treatment.

Transfer to plates with So1E solid medium is followed by a two—day incubation at 30°C. The cells are then harvested and resuspended in 1 ml of G5lb or G51t medium. 100 pl of this bacterial suspension are plated out on a selective SolE medium which, besides kanamycin [25 mg/l] [20 to 35 mg/l] streptomycin Counter- also contains phleomycin and [300 mg/l] as selection of the donor strain selective agents.

[E.coli W3l0l(pME305)] is carried out with the aid of streptomycin. after an colonies this selective So1E medium time of 10 to 14 growing on incubation days which conjugative transconjugants of Sorangium cellulosum have acquired phleomycin resistance owing to transfer of the plasmid pSJB55. These phleomycin- resistant colonies can be used for the subsequent molecular biological investigations. The transformation averages 3 x transfer of 1o'6 frequency for plasmid pSJB55 to Sorangium based on recipient strain SJ3.

The plasmids pSJB5O and pSJB58 can be transferred to Sorangium in an analogous manner. .2 One—stage process .2.1 Cloning of Sorangium DNA into plasmid pSUP2021 The cloning of Sorangium DNA into plasmid pSUP2021 can be carried out as described in Example 5.1.1.

Owing to the helper plasmid pME305 used in 5.1.1 being exchanged for the plasmid pUZ8 [Hedges and Matthew (1979)] which, in contrast to the abovementioned plasmid, carries no ampicillin—resistance gene, the cloning step in the E.coli intermediate host HB101 can be dispensed with because direct cloning in the E.coli donor strain ED8767 which is intended for the conjugal transfer is now possible.

The plasmid pUZ8 is a derivative of the plasmid RP4 which covers a wide host range and is described by Datta et al (1971). The modifications compared with the initial plasmid RP4 relate essentially to the ampicillin—resistance gene and to the insertion element IS21, both of which are deleted, and to the incorporation of an additional gene which confers resistance to mercury ions [see Jaoua et al (1987)]. _34_ The ligation mixture prepared as in Example 5.1.1 can therefore now be transformed directly into the E.coli strain ED8767. For this, competent cells of the E.coli strain ED8767 are prepared with the aid of the processes customarily used for the transformation of E.coli [seez “General recombinant DNA techniques”].

After transformation and subsequent incubation on LB agar supplemented with tetracycline [10 ug/ml] and chloramphenicol [25 pg/ml] for 24 hours, the resulting colonies are subjected to a differential screening by parallel plating out on ampicillin—containing [60 ug/ml] and ampicillin—free medium. It is subsequently possible to isolate those colonies which have lost their ampicillin resistance owing to the integration of the Sorangium DNA fragments. The cultures obtainable in this way can then be employed directly as donor cells for the conjugative transfer of recombinant plasmids into Sorangium cellulosum cells.

In place of the abovementioned ligation mixture, it is, case to clone the pSJB55 or pSJB58, prepared into the E.coli strain ED8767. of course, also possible in this recombinant plasmids pSJB50, as in Example 5.1.1, .2.2 Conjugative transfer of the recombinant plasmids into Sorangium cellulosum For the cellulosum SJ3 actual transfer, 15 ml of a [1—4 x 109 stationary phase are Hdxed with 10 nfl. of a late log Sorangium culture cells/ml] in the phase culture of E.coli donor cells which contain a comparable content of cells. These are then centrifuged together at 4000 rpm for 10 minutes and resuspended in 500 pl of a G51b or G51t medium.

It proves advantageous in this case too for the Sorangium recipient cells to be exposed briefly to a heat treatment with E.coli. in a waterbath before the conjugation The best transfer results can be achieved _35_ with the Sorangium cellulosum strain SJ3 with a heat of 50°C for transfer frequencies of l—5 x treatment at a temperature lO minutes.

Under these conditions, 1o‘5 by a can be achieved, of 10 previous heat treatment. which corresponds to an increase factor compared with a process without Further cultivation of the transformed Sorangium cells is carried out in analogy to the procedure described in Example 5.1.2.

Owing to the use of a restriction—negative E.coli strain as donor strain, such as, for example, E.coli ED8767 [Murray et al (l977)], the transformation frequency can be drastically increased by comparison with the process described above 103). (up to a factor of Molecular genetic analysis (A) Detection of the integration of the plasmid pSJB55 into the chromosome of Sorangium cellulosum SJ3 The DNA which has complete been isolated from the transformed Sorangium cells as described above [compare is digested. with Sml and SalI and loaded [40 mM tris—HCl, 20 mM 2 mM EDTANa2, pH 7.8] After the electrophoresis the placed [l.5 M NaCl, 0.5 M NaOH] for 30 minutes and subsequently in a neutralising solution [l.5 M NaCl, 0.5 M tris—HCl, 1 mM EDTANa2, pH 7.2]. The DNA is transferred, by means of a Southern capillary blotting, buffer Example 4] onto a horizontal tris—acetate sodium [O.9%]. initially in a denaturing solution acetate, agarose gel gel is using a 20-fold concentrated SSC [see section “Media and buffers”] onto a nylon membrane [for example an Amersham Hybond nylon membrane; Amersham. International plc, Amershanl Place, Amersham, England HP7 9NA] and fixed there by UV treatment for 6 minutes. Further details of this process are described in the Amersham International _36_ handbook (1985).

“Membrane Transfer and Detection Methods”, The DNA intended as hybridisation probe is labelled by means of a nick translation [Rigby DWJ et al (l977)].

This the form of the ”P—labelled Pvul insert comprising 3.5 Kb from the plasmid pSJB55. The actual takes out of Denhardt hybridisation is carried using a slight modification of the process [Denhardt DT (1976)]. The buffer used for the prehybridisation and hybridisation has the 6 x SSC (l982)] [Maniatis et al + 0.5% SDS + 0.2 mg/ml denatured salmon sperm following composition: [Maniatis et a1 + 5 x Denhardt (l982)] DNA.

The prehybridisation is carried out at 65°C and takes 3 the reaction is P__ hours, while actual hybridisation complete after 20 hours. For the hybridisation, a labelled [lO5 cpm per cm2 of filter], denatured Pvul fragment comprising 3.5 Kb from plasmid pSJB55 is added. After the hybridisation the filter is washed first for 2 X 15 minutes in 2-fold concentrated SSC at a temperature of 65°C, subsequently in 2 x SSC + 0.1% SDS likewise at 65°C for 30 nunutes and finally once more in 0.5 x SSC [15 minutes at 65°C].

The subsequent autoradiography is carried out using an film FUJY X film]. The autoradiographs show no bands X—ray [for example Rays of the transconjugants which correspond to free pSJB55 plasmid DNA which is in over—spiralised form. By contrast, however, a positive signal is found in the chromosomal region of the filter membrane. bands of The hybridisation pattern of the The SmaI—digested plasmid pSJB55 provides 8.9,6.7 and 1.6 Kb. parent strain SJ3 after Smal digestion likewise shows three bands, one of which corresponds to an internal fragment, comprising 1.6 Kb, of the Sorangium insert -37.. cloned into the plasmid pSJB55. The pattern of the SmaI—digested DNA of the transconjugants [8.9 and 6.7 Kb band of plasmid pSJB55 .6 Kb band which is hybridisation shows 5 bands and SJ3 bands] common to all three. including the After Sall digestion, plasmid pSJB55 (the other Sall fragment comprising 3.1 Kb of pSJB55 does not hybridise with the probe). The the SalI—digested SJ3 DNA a band of 14.1 Kb is found for hybridisation pattern of likewise shows a band of 5 Kb. In the transconjugants the 14.1 Kb fragment of plasmid. pSJB55 and the 5 Kb fragment of SJ3 disappear after Sall digestion.

These are replaced by two new bands of 11.5 Kb and 7.7 The Smal data show that all the pSJB55 fragments are intact in the genome of the transconjugants. This rules out the possibility of a site—specific recombination because in this case at least one of the Smal fragments would have had to disappear. the of the Sall digestion make it clear that the Furthermore, results plasmid pSJB55 has been integrated into the Sorangium genome, specifically at the site where the DNA region homologous with pSJB55 is located (= 3.5 Kb Pvul fragment). This integration takes place in the course of a homologous recombination between a Sorangium insert comprising 3.5 Kb from pSJB55 and the same insert within the Sorangium genome.

MEDIA AND BUFFER SOLUTIONS G51b medium (pH 7.4) Glucose 0.2% Starch 0.5% [potato starch, Italy] [DIFCO Laboratories, Noredux type; CERESTAR ITALIA S.p.a., Milan, Peptone USA] Q C) |—‘ (\) o\0 o\0 Probion S _38_ [Single Cell Protein; HOCHST AG, Frankfurt, FRG] CaCl2 X 2 H20 0.05% MgS04 x 7 H20 0.05% HEPES [FLUKA] 1.2% G51t medium (pH 7.4) Glucose 0.2% Starch 0.5% [potato starch, Noredux type; CERESTAR ITALIA S.p.a., Milan, Italy] Tryptone [MARCO, Hackensack, NJ USA] 0.2% Probion S 0.1% [Single Cell Protein; HOCHST AG, Frankfurt, FRG] CaCl2 X 2 H20 0.05% MgS04 x 7 H20 0.05% HEPES [FLUKA] l. % G52c medium (pH 7.4) Glucose 2.0 g/l Starch 8.0 g/l [potato starch, Noredux type; CERESTAR ITALIA S.p.a., Milan, Italy] Soya meal defatted 2.0 g/l [MUCEDOLA S.r.l., Settimo Milanese, Italy] Yeast extract 2.0 g/l [FOULD & SPRINGER, Maison Alfort, France] CaCl2 X 2 H20 1.0 g/l MgS04 x 7 H20 1.0 g/l Fe-EDTA (8 g/l stock solution] 1.0 ml HEPES [FLUKA] 2.0 g/l Distilled water ad 1000 ml pH is [20 minutes at 120°C]. adjusted to 7.4 with NaOH before sterilisation pH after sterilisation: 7.4 SolE medium (pH 7.4) Glucose* 0.35% Tryptone [MARCO, Hackensack, NJ USA] 0.05% MgS04 X 7 H20 0.15% Ammonium sulphate* CaCl2 X 2 H2O* K2HPO4* Sodium dithionite* Fe—EDTA* HEPES [FLUKA] Supernatant of a sterilised, stationary S. cellulosum culture Agar l—'C)©CD©© U707 o\0 .05% .l% .006% .0l% .O0O8% (V/V) *Addition takes place only after sterilisation pH is adjusted to 7.4 with NaOH before sterilisation [20 minutes at 120°C].

LB medium Tryptone Yeast extract NaCl STE buffer (pH 8.0) Sucrose EDTANa2 Tris~HCl RLM buffer (pH 7.6) SDS EDTANa2 Tris—HCl TER buffer Tris-HCl (pH 8.0) 1 mM EDTANa2 RNAse Ligation buffer Mgclz Tris—HCl (pH 7.8) .0 g/1 .0 g/l .0 g/l % 1 mM l0 mM % l25 mM 0.5 mM mM 1 mM pg/ml .1 M 0.5 M TABLES Table 1: Bacterial strains and plasmids Strain Relevant characteristics Escherichia coli W310lNal HBlOl ED8767 Sorangium cellulosum So Ce 26 So ce 26/SJ3 RecAl3, trpE, NalR F—, hsds2O (r-, m—), recAl3, ara 14, proA2, lacYl, galK2, rpsL20 (SmR), xyl—5, mtl—l, sup E 44, lambda- recA, supE, supF, hsds Wild-type strain SmR spontaneous mutant Plasmid pSUP202l Ap, Cm, Km, Ph pSJB5O Cm, Km, Ph pSJB55 Cm, Km, Ph pSJB58 Cm, Km, Ph pME305 Ap, Tc pUZ8 Tc, Km, Hg DEPOSITION Within the scope of the present application, the following microorganisms and plasmids have been deposited at the “Deutsche Sammlung Von Mikroorganismen und Zellkulturen GmbH” in Braunschweig (FRG), which is recognised in accordance with the Budapest Treaty as international depository, to comply with the requirements for the international recognition of the deposit of microorganisms for the purpose of patenting.

Microorganism/ Date of Deposit Date of viability plasmid deposit number certificate pSJB55 25.01.1991 DSM 6321 25.01.1991 (cloned into E.coli) Sorangium 25.01.1991 DSM 6380 14.02.1991 cellulosum So ce 26/SJ3 LIST OF REFERENCES Datta N et al, J Bacteriol 108: 1244-1249 (1971) Denhardt DT, Biochem Biophys Res Commun, 23: 641-646 (1976) Hedges RW and Matthew M, Plasmid 2: 269-278 (1979) Gentz R et al, Proc Natl Acad Sci, USA 18: 4926-4940 (1981) Jaoua S et al, Plasmid 18: 111-1l9(1987) Jaoua S et al, Plasmid 13: 183-193(1990) Kaiser D, Genetics of Myxobacteria, in: “Myxobacteria: Development and Cell Interactions”, ed E Rosenberg, pp 163-184, Springer Verlag, Berlin/New York (1984) Kuner JM and Kaiser D, Proc Natl Acad Sci USA, 18:425- 429 (1981) Kuspa A and Kaiser D, J Bacteriol 111: 2762-2772 (1989) Maniatis T et al, “Molecular Cloning”, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) Miller JH, “Experiments in Molecular Genetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972) Murray NE et al, Mol Gen Genet 150: 53 (1977) O’Conner KA and Zusman DR, J Bacteriol 155: 317-329 (1983) ' Rella M, Dissertation ETH Zurich, No. 7601, SEITZ Reichenbach H et al, Trends in Biotechnology, 6: 115- 121 (1988) Rigby DWJ et al, J Mol Biol, 113: 237-251 (1977) Rosenberg M and Court D, Ann Rev Genetics 13: 319-353 (1979) Shimkets LJ and Asher SJ, Mol Gen Genet 111: 63-71 (1988) Shimkets LJ et al, Proc Natl Acad Sci USA 80: 1406- 1410(1983) Simon R et al Bio/Technol, November 1983: 784-791 (1983) EP 0 310 619 EP 0 358 606 EP 0 372 230 US-P 4,910,140

Claims (30)

PATENT CLAIMS

1. Process for the genetic manipulation of myxobacteria from the Sorangium/Polyangium group, characterised in that (a) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin, which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (b) genetic material which naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the artificially linked with the and is therefore aid of myxobacterial chromosome rDNA techniques known per se DNA to such homologous or else essentially result of which the flanked by homologous or else essentially homologous DNA sections; the cell recombination, at a homologous sections, as a genetic material to be inserted is said is inserted into myxobacterial and there integrated, via homologous site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the bacteria, into the chromosome of said myxobacteria.

2. Process according to Claim l, characterised in that said genetic material to be inserted is an expressable DNA which is linked in an operable manner to expression sequences able to function in the myxobacterial cell.

3. Process according to Claim 1, characterised in that the insertion of the genetic material takes place via a donor organism with capable of the conjugation—like information exchange myxobacterium target organism from the donor to the myxobacterium recipient. the the

4. Process for genetic manipulation of myxobacteria from Sorangium/Polyangium group according to Claim 1, which is characterised by the following process steps: the DNA of the myxobacterium target organism or of a related organism; (a) preparation of complete (b) fragmentation of the complete DNA isolated as in (a); (c) cloning of the fragments as prepared in (b) into suitable plasmid vectors and transformation of said vectors into one of the host organisms normally used for cloning purposes; (d) selection of those colonies which contain a plasmid with integrated myxobacterium DNA fragment and isolation of said plasmids; (e) transformation of the plasmid DNA isolated as in (d) into a donor organism capable of conjugation—like information exchange with the myxobacterium target organism; (f) conjugal transfer of the recombinant plasmid DNA to the myxobacterium target organism; (g) cultivation of the transformed myxobacterium cells and selection of positive transformants.

5. Process according to either of Claims that the l or 4, characterised in genetic material to be inserted. is a plasmid. which may contain one or more expressable DNA sections and which contains the homologous or else the essentially homologous DNA regions cloned in.

6. Process according to either of Claims 1 or 4, characterised in that the degree of homology between said homologous DNA sections and the corresponding regions within the myxobacterial chromosome is between 80% and 100%.

7. Process according to that either of Claims DNA 1 or 4, characterised in said homologous sections comprise at least 100 Bp. either of Claims 1 or 4, DNA

8. Process according to characterised in that said homologous sections comprise between 0.3 and 4 Kb.

9. Process according to Claim 4, characterised in that a prokaryote selected from the group consisting of E. coli, pseudomonads, actinomycetes, salmonellae and myxobacteria themselves is used as host organism for the cloning of the myxobacterium DNA fragments or as donor organism: within the scope of the conjugal DNA transfer.

‘ 10. Process according to Claim 4, characterised in that a restriction—negative or restriction-deficient bacterial strain is used as host organism for the cloning of the myxobacterium DNA fragments or as donor organism within the scope of the conjugal DNA transfer.

11. Process according to Claim 10, characterised in that the cloning of the myxobacterium DNA fragments is carried out directly in a donor organism which is capable of conjugation-like information exchange with the myxobacterium target organism.

12. Process according to Claim 4, characterised in that the myxobacterium cells are subjected to a brief heat treatment immediately before the conjugal DNA transfer.

13. Process according to Claim 12, characterised in that the heat treatment is carried out at a temperature of 35°C to 60°C over a period of 1 to 120 minutes.

14. Recombinant DNA molecule which makes possible integration of genetic material at a defined site within the genome of myxobacteria from the Sorangium/Polyangium group, characterised in that said recombinant DNA molecule contains the DNA which is to be integrated, and in that said DNA has homologies with corresponding DNA regions within the myxobacterial genome, or else is flanked by one or more such homologous DNA sequences, to an extent such that, on transformation of the myxobacterial cell containing the homologous DNA region, there is integration of said DNA, which is to be integrated, at a site, which is exactly defined by reason of the homology present between the inserted DNA and the DNA intrinsic to the within bacteria, the myxobacterial genome via homologous recombination.

15. Recombinant DNA molecule according to Claim 14, characterised. in that the degree of homology between DNA the regions within the myxobacterial chromosome is between 80 and 100%.

16. Recombinant DNA molecule according to Claim 14, that DNA said homologous sections and corresponding characterised in sections said homologous comprise a region of at least 100 Bp.

17. Recombinant DNA molecule according to Claim 14, characterised in that said DNA itself with sequence to be has DNA integrated already sufficiently great the DNA homology corresponding regions within bacterial genome so that direct exchange of this sequence for said homologous genomic DNA can take place by means of homologous recombination.

18. Recombinant DNA molecule according to Claim 14, characterised in that said DNA to be double—stranded DNA.

19. Recombinant DNA molecule according inserted is to Claim 14, characterised in that said DNA to be inserted is single—stranded DNA.

20. Recombinant DNA molecule according to Claim 14, characterised in that said DNA to be inserted is an expressable DNA which is linked in an operable manner to expression sequences able to function in the myxobacterial cell.

21. Recombinant DNA molecule according to Claim 14, characterised in that said flanking DNA sections are fused together to a unit, as component of a DNA molecule which is closed in the form of a ring.

22. Recombinant DNA molecule according to Claim 14, characterised in that said homologous DNA sections originate from the myxobacterium genome itself.

23. Cloning vector containing a recombinant DNA molecule according to any of Claims 14 to 22.

24. Plasmid DNA for the conjugal transfer from a donor organism to the myxobacterium recipient from the Sorangium/Polyangium group, characterised in that said plasmid DNA contains, besides the DNA to be inserted, homologous or else essentially homologous DNA sections, and transfer (tra) and mobilisation functions (mob) suitable for transfer into myxobacterium cells.

25. Plasmid DNA according to Claim 24 containing a recombinant DNA molecule according to any of Claims 14 to 22.

26. Process for the preparation of a recombinant DNA Inolecule according to Claint 14, characterised in that the DNA which is to be integrated, which has homologies with corresponding DNA regions within the myxobacterial genome to an extent such that, on transformation of the myxobacterial genome containing the said homologous DNA region, said DNA at a site, there is integration of which is defined by reason of the homology present between the inserted DNA and the DNA intrinsic to the bacteria, within the bacterial genome via homologous recombination, (a) is isolated from a source which contains homologous or else essentially homologous DNA sections; or (b) when said DNA which is to be integrated naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome, this DNA is artificially linked with the aid of rDNA techniques known per se to corresponding homologous or else essentially homologous DNA sections.

27. Process according to Claim 26, characterised in that said DNA to be inserted is located on a plasmid, and the homologous DNA sequences are cloned into the plasmid DNA at any desired site without, however, thereby destroying the functional integrity of the DNA to be inserted.

28. Process for the preparation of the characterised in that genetically modified myxobacteria from Sorangium/Polyangium group according to Claim 1, (a1) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (a2) genetic material which naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome and is therefore artificially linked with the aid of rDNA techniques known per se to such homologous or else essentially homologous DNA sections, as a result of which the genetic material to be inserted is flanked by said homologous or else the essentially homologous DNA sections; is inserted into the myxobacterial cell and there integrated, via homologous recombination, at a site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the the chromosome of said bacteria, into myxobacteria; and (b) positive transformants are selected with the aid of selection processes known per se and cultivated as pure culture. cell prepared by a process according to any of Claims l to 13 and 28.

29. Genetically modified myxobacterial

30. Genetically modified myxobacterial cell from the Sorangium/Polyangium group containing an exogenous DNA of homologous and/or heterologous origin integrated into the myxobacterial via genome homologous recombination. F. R. KELLY & co., AGENTS FOR THE APPLICANTS.