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\IIIIIIIIIIIIIIllIlllllllllIllll||l||l||llllllillllllllllllllllllllllllliUS005686295A United States Patent [191 [11] Patent Number: 5,686,295 Jaoua et al. [45] Date of Patent: Nov. 11, 1997

[54] PROCESS FOR THE GENETIC Denhardt, Biochem. Biophys. Res. Comm, 23:641-646 MANIPULATION OF MYXOBACTERIA (1976). Hedges et al., Plasmid, 2:269-278 (1979). ['75] Inventors: Samir Jaoua. Villa Mounir Sfax. Gentz et al.. PNAS USA. 78:4926-4940 (1981). Tunisia; Thomas Schupp. Mohlin; Jaoua et al.. Plasmid. 18:111-119 (1987). Snezana Neli‘. Bubendorf. both of Jaoua et al.. Plasmid. 23:183-193 (1990). Switzerland Kaiser, Genetics of Myxobacteria, in: Myxobacten'a: Devel [73] Assignee: Novar?s Finance Corporation. New opment and Cell Interactions, ed. by E. Rosenberg. (Berlin! York, NY. New YorkzSpringer Verlag, 1984), pp. 163-184. Kuner et al.. PNAS USA. 78:425-429 (1981). Kuspa et al.. J. Bacteriol. 171:2762-2772 (1989). [21] Appl. No.: 276,752 Maniatis et al.. Molecular Cloning. New York:Cold Spring [22] Filed: Jul. 18, 1994 Harbor Laboratory. 1982. Miller. Eipen'ments in Molecular Genetics. New York: Cold Related US. Application Data Spring Harbor Laboratory. 1972. Min-ray et 111.. Mol. Gen. Genet, 150:53 (1977). [63] Continuation of Ser. No. 841,680, Feb. 26, 1992, abandoned. O’Conner et al.. J. BacterioL. 155:317-329 (1983). [30] Foreign Application Priority Data Rella. Dissertation ETH Zurich. No. 7601, SFH‘Z. Reichenbach et :21. Trends in Biotechnology. 6:115-121 Mar. 1, 1991 [CH] Switzerland ...... 626/91 ( 1988). [51] Im. CLG ...... C12N 1/21;c12N15/63 Rigby et al.. J. Mol. BioL. 113:237-251 (1977).

[52] US. (:1...... 435/2523; 435/320.1; Rosenberg et 31.. Ann. Rev. Genetics. 13:319-353 (1979). 536/231; 536/237; 536/241 Shimkets et al.. PNAS USA. 80:1406-1410 (1983). [58] Field of Search ...... 435/691. 172.1. Simon et al.. Bio/TechnoL. 784-791 (Nov. 1983). 435/1723. 252.3. 320.1; 536/23.1. 23.7. Shimkets et al.. Mol. Gen. Genet. 211: 63-71 (1988). 24.1 Primary Examiner-David Guzo [56] References Cited Attorney, Agent, or Firm-—l. Timothy Meigs U.S. PATENT DOCUMENTS [57] ABSTRACT

4,910,140 3/1990 Dower ...... 435/1723 The present invention relates to a novel process for the genetic manipulation of myxobacteria. preferably of myxo FOREIGN PATENT DOCUMENTS bacteria of the Sorangium/Polyangium group. which makes 0317509 5/1989 European Pat. Off. . it possible for the ?rst time speci?cally to apply recombinant 0358606 3/1990 European Pat. Off. . DNA techniques to this group of organisms. The technical 0372230 6/1990 European Pat. Off. . implementation of this process is based primarily on the WO87/07909 12/1987 WIPO . preparation of recombinant DNA molecules which. by rea son of their speci?c construction. are able to integrate genes OTHER PUBLICATIONS or DNA sequences which code. where appropriate. for novel Norton. “Microbiology” 2nd Ed. Addison-Wesley Pub. Co. and desirable properties. with the aid of homologous recom 1986. pp. 253-255. bination at sites. which are accurately de?ned by reason of Breton et al.. J. BacterioL. 161:523-528 (1985). the homologies present. within the bacterial genome. and on Breton et al.. J. BiotechnoL. 4303-311 (1986). the insertion thereof into the myxobacterial cell. Breton et al.. FEMS Microbiol. Lett.. 40:183-188 (1987). Datta et al.. J. BacterioL. 198:1244-1249 (1981). 12 Claims, No Drawings 5,686,295 1 2 PROCESS FOR THE GENETIC Another process for gene transfer is based on the use of MANIPULATION OF MYXOBACTERIA the plasmid RP4 which has a very wide host range. Breton et al (1985) were able to show that this plasmid can be This application is a continuation of application Ser. No. transferred via conjugation from E. coli into Myxococcus 07/84l.680. ?led Feb. 26. 1992. now abandoned. xanthus, and is there stably integrated into the chromosome. The present invention relates to a novel process for the Based on these properties. Breton et al (1986) and Breton genetic manipulation of myxobacteria. preferably of myxo and Guespin-Michel (1987) were able to integrate foreign bacteria of the Sorangium/Polyangium group. which makes genes into the chromosome of Myxococcus xanthus. Inves it possible for the ?rst time speci?cally to apply recombinant tigations by Jaoua et a1 (1987; 1989) revealed that the DNA techniques to this group of organisms. 10 observed integration is based. with a high degee of In particular, the present invention relates to a process for probability. on a so-called site speci?c recombination. The the insertion of DNA sequences of homologous or heterolo latter is con?ned to particular sites. which have a narrow spatial restriction. Within the Myxococcus xanthus chromo gous origin or a combination of DNA sequences of homolo some and is mediated by one or more so-called hot spots on gous or heterologous origin into the chromosome of said the RP4 plasmid. In addition. it has emerged during the myxobacteria via homologous recombination. and to geneti 15 investigations carried out within the scope of the present cally modi?ed myxobacteria prepared with the aid of this invention that the previously known Myxococcus system process. discovered here cannot be applied to bacteria of the Likewise embraced are recombinant DNA molecules. Sorangium/Polyangium group. It is assumed that these plasmids and vectors which are particularly suited for use in organisms lack the speci?c structural elements which are the process according to the invention. and genetically necessary for site speci?c recombination on their chromo modi?ed myxobacteria of the Sorangium/Polyangium group somes. In addition. it has been found that no stable trans containing exogenous DNA of homologous and/or heterolo position takes place with these organisms either. for example gous origin. on use of transposon Tn5. The myxobacteria of the Sorangium/Polyangium group The object which it was intended to achieve within the are highly specialised organisms which are commonly scope of this invention thus related primarily to the provi detectable in soil samples. dead plant material or in animal sion of a universally applicable process for the genetic dung. Characteristic of this group of microorganisms is their manipulation of all myxobacteria. but especially of myxo ability to utilise cellulose or cellulose-containing degrada bacteria of the Sorangium/Polyangium group. which is free tion products as sole carbon source. Another characteristic of the abovementioned restrictions of the known processes feature of this group is their ability to produce highly active and thus permits undirected or else. preferably. targeted secondary metabolites. insertion of genetic material into myxobacteria. independent A large number of strains from this group which. for of structural elements present on the myxobacterial chro example. are able to synthesise plant-microbicidal com mosome or of speci?c transposition events. pounds have now been described. Particularly important in This can be achieved according to the invention. for this connection are the so-called soraphens. macrocyclic 35 example. by preparing genetic constructs. especially plas compounds which have a bene?cial biocidal spectrum mid vectors. which. by reason of their speci?c structure. can against phytopathogenic microorganisms. but especially be inserted at the desired sites within the chromosome and against phytopathogenic fungi. These compounds have very which are not linked. as is the case in the previously known advantageous curative. systemic and. in particular. preven processes. to particular integration sites predetermined by tive properties and can be employed to protect numerous the functional organisation of the myxobacterium chromo crop plants [EP 0 358 606]. some or of the plasmid used (hot spots). or else dependent It is also known of other representatives of the group of on transpositions of integrated transposons. myxobacteria that they are able to synthesise highly active The present invention thus relates primarily to a process compounds with antibiotic potency [Reichenbach et al for the genetic manipulation of bacteria of the order (1988)]. Because of the importance of these compounds. 45 Myxobacterales. but especially of myxobacteria of the there is a great interest in understanding the genetic bases of Sorangium/Polyangium group. which is characterised in that their synthesis in order thus to provide the possibility of genetic material of homologous or heterologous origin or a being able speci?cally to in?uence these where appropriate. combination of genetic material of homologous and heter The precondition for this is the provision of a process ologous origin is inserted into the myxobacterial cell and which makes possible direct. and preferably targeted. 50 integrated. via homologous recombination at random or else. manipulation of these organisms using recombinant DNA when there is appropriate knowledge of the structural and techniques. for example by the targeted incorporation of functional organisation of the bacterial genome. speci?cally novel genes or gene fragments or other DNA sequences, at a site. which is accurately de?ned on the basis of the including whole plasmids. into the genome of the myxobac homology present between the inserted DNA and DNA teria. 55 intrinsic to the myxobacteria. into the chromosome of said A few representatives of the group of myxobacteria have myxobacteria. independent of structural elements present on already been the subject of investigations in this direction. the myxobacterial chromosome or of speci?c transposition Special interest in this connection was primarily directed at events. Myxococcus xam‘hus, which is a myxobacterium which has The process according to the invention for the genetic now been extensively researched and for which various gene manipulation of bacteria of the order Myxobacterales is transfer processes have already been described. Thus. for particularly characterised in that example. the coli phage Pl has been used very intensively. (a) genetic material of homologous or heterologous ori initially for the insertion of transposon TnS into the Myxo gin or a combination of genetic material of homologous and coccus xanthus chromosome [Kaiser (1984); Kuner and heterologous origin. which naturally contains one or more Kaiser (1981)] and then later for the transfer of genes cloned 65 DNA sections which are homologous with or else at least in Myxococcus xanthus back into the original E. coli host essentially homologous with a corresponding region on the [O’Conner and Zusman (1983); Shirnkets et al (1983)]. myxobacterial chromosome; or else 5,686,295 3 4 (b) genetic material which naturally contains no sections Besides the targeted and thus very e?icient in situ modi which are homologous with or else at least essentially ?cation of genes intrisic to the bacteria (directed homologous with a corresponding region on the myxobac mutagenesis). the process according to the invention has a terial chromosome and which is therefore arti?cially linked number of other possible applications such as. for example. to such homologous or else essentially homologous DNA the incorporation of additional gene copies in regions known sections using rDNA techniques known per se; is inserted to have a high expression rate. and elimination and thus into the myxobacterial cell and there integrated. via homolo switching off of unwanted genes. It is furthermore also gous recombination, at a site. which is accurately de?ned by possible now to think about the following further possible reason of the homologies present between inserted DNA and applications. DNA which is intrinsic to the bacteria. into the chromosome Incorporation of strong or controllable promoters in front of said myxobactmia. independent of structural elements of genes. intrinsic to the bacteria. with interesting present on the myxobacterial chromosome or of speci?c functions. transposition events. Cloning of genes of various origin into myxobacteria. but The integration of the genetic material into the bacterial especially into myxobacteria of the Sorangium/ chromosome via homologous recombination is mediated by Polyangium group. one or more DNA sections within the DNA to be inserted. Cloning and expression of genes of various origin. which are homologous with or else at least essentially homologous with a corresponding region on the myxobac Transfer of the inserted DNA back into another microor terial chromosome. It is thus not bound to the presence of ganism such as. for example. into E. coli for further particular chromosomal structures and can in principle take processing. place at any desired site within the bacterial chromosome. Use of the heterologous DNA of an inserted vector ?rstly The homologous DNA sections which can be used within as radioactive probe for identifying the myxobacterium the scope of the process according to the invention do not fragments adjacent to the site of integration. and sec necessarily have to have 100% identity with the correspond ondly as pattern [template] for ampli?cation within the ing sections on the myxobacterial chromosome in order to 25 scope of a polymerase chain reaction [PCR]. be able to bring about the required recombination event. On The present invention further relates to a process for the the contrary. it su?'ices for these DNA sections to be essen preparation of genetically modi?ed bacteria of the order tially homologous with the corresponding regions on the Myxobacterales. but especially of bacteria of the bacterial genome. that is to say when these have a degree of Sorangium/Polyangium group. which is characterised in that homology of between 60% and 100%. preferably between (a 1) genetic material of homologous or heterologous 80% and 100% and very particularly preferably between origin or a combination of genetic material of homologous 90% and 100%. and heterologous origin. which naturally contains one or Thus. “homologous” DNA sections are intended also to more DNA sections which are homologous with or else at mean hereinafter those sections which do not have 100% least essentially homologous with a corresponding region on identity with the corresponding regions on the myxobacte 35 the myxobacterial chromosome; or else rial chromosome but are at least “essentially homologous” (a2) genetic material which naturally contains no sections therewith. which are homologous with or else at least essentially It is possible in this connection for the homologous DNA homologous with a corresponding region on the myxobac sections to be isolated either from the target organism itself terial chromosome and which is therefore arti?cially linked or else from related organisms. for example after fragmen 40 to such homologous or else essentially homologous DNA tation of the particular genome. When the DNA sequence of sections unsing rDNA techniques known per se; is insmted the DNA sections on the myxobacterial chromosome which into the myxobacterial cell and there integrated. via homolo are intended in each case to be used for integration is known. gous recombination. at a site. which is accurately de?ned by the corresponding DNA fragments which are homologous reason of the homologies present between inserted DNA and with or else at least essentially homologous with these 45 DNA which is intrinsic to the bacteria. into the chromosome chromosomal sections can. of course. also be prepared by of said myxobacteria. independent of structural elements synthesis. present on the myxobacterial chromosome or of speci?c The DNA sections of homologous origin which can be transposition events; and used within the scope of the present invention can moreover (b) positive transforrnants are selected with the aid of be either DNA sections of known sequence or else DNA 50 selection processes known per se and cultivated as pure fragments obtainable at random. for example after restric culture. tion digestion of homologous DNA. The present invention thus makes possible for the ?rst When the structural and functional organisation of the time a targeted genetic manipulation of the genome of appropriate parts of the myxobacterial genome is known. the myxobacteria. but especially of myxobacteria of the process according to the invention thus makes possible for Sorangium/Polyangium group. where the integration of the the ?rst time a targeted. predictable modi?cation of the genetic material can be mediated. depending on the particu genes present on the myxobacterial genome (in situ lar of aim of the planned procedure. alternatively either by modi?cation) by exchange of natural or arti?cially modi?ed homologous DNA sections of known sequence or else by genes. gene fragments or other useful DNA sequences with randomly selected homologous sections of unknown homologous DNA sections within the bacterial genome. The sequence. process according to the invention furthermore makes pos The present invention likewise embraces recombinant sible a speci?c identi?cation of the function of individual DNA molecules which. by reason of their speci?c genes within the complete genome of myxobacteria by construction. are able to integrate genetic material such as. speci?c switching off of genes [gene disruption] or by for example. genes or gene fragments or other useful DNA complementation of genes which have previously been 65 sequences which. where appropriate. cede for novel and inactivated by application of other processes. especially of desirable properties. with the aid of homologous recombi mutation processes. nation at random or else. when the structural and functional 5,686,295 5 6 organisation of the bacterial genome is known, also targeted Mutants. variants: Spontaneously or else arti?cially. by at sites. which are accurately de?ned by reason of the application of known process measures such as, for homologies present between inserted DNA and DNA intrin example. UV treatment, treatment with mutagenic agents sic to the bacteria. within the bacterial genome. as well as etc.. produced derivative of a microorganism which still has processes for the preparation of said recombinant DNA 5 the features and properties. essential to the invention. of the molecules. initial strain which has acquired the latter by reason of the The present invention furthermore embraces genetically transformation with exogenous DNA. modi?ed myxobacteria. especially modi?ed myxobacteria It has now been possible for the ?rst time within the scope of the Sorangiurn/Polyangium group with, where of the present invention to provide a process which makes appropriate. novel and/or improved properties. which have 10 possible a preferably targeted genetic manipulation of the been prepared by insertion of said recombinant DNA mol genome of bacteria of the order Myxobacterales. but espe ecules. cially of bacteria of the Sorangium/Polyangium group. in The present invention additionally relates to the offspring that it is now possible for genetic material to be integrated. of said modi?ed myxobacteria and to mutants and variants and also to be expressed therein. at random or else. when thereof which still contain said recombinant DNA molecule. there is appropriate knowledge of the structural and func A number of terms which are customary in recombinant tional organisation of the bacterial genome. speci?cally at DNA technology and in bacterial genetics are used in the accurately defined positions. which can be predetermined in following description. some cases. within the bacterial genome. independent of In order to ensure clear and uniform understanding of the structural elements present on the myxobacterial chromo description and of the claims. as well as of the scope 20 some or of speci?c transposition events. intended to apply to said terms. the following de?nitions are The process according to the invention is. moreover. stated: essentially based on the recognition that it is possible to Gene(s) or DNA of heterologous origin: A DNA sequence incorporate exogenous genetic material with the aid of which codes for a speci?c product or products or ful?ls a homologous recombination into the genome of biological function and which originates from a species 25 myxobacteria. it being possible to use. besides natural. also other than that into which the said gene is inserted; said arti?cally modi?ed and/or synthetic genes or gene fragments DNA sequence is also called foreign gene or foreign DNA. or other DNA sequences including whole plasmids. as long or exogenous DNA. as they comprise DNA sections or are ?anked by DNA Gene(s) or DNA of homologous origin: A DNA sequence sections which have a homology. which is su?icient for which codes for a speci?c product or products or ful?ls a 30 recombination. with corresponding sections on the myxo biological function and which originates from the same bacterial genome. species into the which the said gene is inserted. This DNA The homologous DNA sections which can be used within is also called exogenous DNA. the scope of the present invention do not in this connection DNA homology: Degree of agreement between two or necessarily have to have 100% identity with the correspond more DNA sequences. 35 ing sections on the myxobacterial chromosome in order to Synthetic gene(s) or DNA: A DNA sequence which codes be able to bring about the required recombination event. On for a speci?c product or products or ful?ls a biological the contrary. it suffices if these DNA sections are essentially function and which is prepared by a synthetic route. homologous with the corresponding regions on the bacterial Promoter: A control sequence of DNA expression which genome. that is to say if these have a degree of homology of ensures the transcription of any desired homologous or between 60% and 100%. preferably between 80% and 100% heterologous DNA gene sequence in a host cell. as long as and very particularly preferably between 90% and 100%. said gene sequence is linked in an operable manner to a ‘The size of said homologous regions can vary. but ought promoter of this type and the latter is active in said host cell. to be at least 100 Bp. Regions of homology which comprise Termination sequence: DNA sequence at the end of a between 0.3 Kb and 4 Kb. but preferably between 1 Kb and transcription unit which signals the end of the transcription 45 3 Kb. are preferred within the scope of this invention. process. Recombinant DNA molecules which. by reason of their Overproducing promoter (OPP): Promoter which is able speci?c construction. make possible the speci?c incorpora to bring about in a host cell the expression of any functional tion of genes or gene fragments or other interesting DNA gene sequence(s) linked in an operable manna to an extent sequences. including whole plasmids. into the genome of a (measured in the form of the RNA or of the polypeptide 50 target cell with the aid of homologous recombination in the amount) which is distinctly higher than is naturally observed abovementioned manner form an essential component of the in host cells which are not transformed with said OPP. present invention. 3'15‘ non-translated region: DNA sections which are The present invention relates in particular to recombinant located downstream/upstream of the coding region and DNA molecules which make possible a targeted integration which. although transcribed into mRNA. are not translated 55 of genetic material such as. for example. genes. gene frag into a polypeptide. This region contains regulatory ments or other DNA fragments at a de?ned site within the sequences such as. for example. the ribosome binding site genome of myxobacteria. but especially of myxobacteria of (5'). the Sorangium/Polyangium group. and which are character DNA expression vector: Cloning vehicle such as. for ised in that they contain the DNA which is to be integrated. example. a plasmid or a bacteriophage. which contains all and in that said DNA has homologies with corresponding signal sequences which are necessary for the expression of DNA regions within the myxobacterial genome. or else is an inserted DNA in a suitable host cell. ?anked by such homologous DNA sequences. to an extent DNA transfer vector: Transfer vehicle such as. for such that. on transformation of the myxobacterial cell con example. a plasmid or a bacteriophage vector. which makes taining the homologous DNA region. there is undirected or it possible to insert genetic material into a suitable host cell. 65 else. preferably. targeted integration of said DNA. which is Homologous recombination: Reciprocal exchange of to be integrated. at a site. which is exactly de?ned by reason DNA pieces between homologous DNA molecules. of the homology present between the inserted DNA and the 5 ,686.295 7 8 DNA intrinsic to the bacteria. within the myxobacteria] Every promoter and every terminator which is able to genome via homologous recombination, independent of bring about induction of expression of an expressible DNA structural elements present on the myxobacterial chromo sequence in myxobacteria can be used as component of the some or of speci?c transposition events. hybrid gene construction. Said recombinant DNA molecules can be prepared very Examples of promoters suitable for use in the process straightforwardly in such a way that the DNA which is to be according to the invention are integrated and which has the abovementioned properties the light-inducible promoter of Myxococcus xanthus [EP (a) is isolated from a suitable source; or 310 619]; (b) when said DNA which is to be integrated naturally other Myxococcus mnthus promoters; contains no sections which are homologous with or else at 10 promoters of Actinomycetes. especially of Strepto least essentially homologous with a corresponding region on rnycetes; the myxobacterial chromosome. this DNA is arti?cially E. coli promoters such as. for example. the Tac(hybrid), linked with the aid of rDNA techniques known per se to PL or Trp promoter. corresponding homologous or else essentially homologous Suitable termination sequences which can be used within DNA sections. the scope of this invention are described. for example. by If the DNA which is to be integrated is an expressible Rosenberg and Court (1979) and by Gentz et a1 (1981). DNA sequence it is advantageous for the latter to be linked The functional unit which has been formed in this way in an operable manner to expression signals capable of and consists of a gene and of expression signals active in functioning in the bacterial cell. and. where appropriate. to myxobacterial cells can subsequently. where appropriate. be be ?anked by DNA sections which have homologies with a 20 ?anked by one or more DNA sections which have homolo particular region within the bacterial genome. These gies with corresponding DNA regions within the myxobac ?anking. homologous DNA sections are present. preferably terial genome to an extent such that. on transformation of the fused together to a unit. as component of DNA molecules myxobacterial cell containing said homologous region. there which are closed in the form of a ring. is random or else. preferably. speci?c integration of a gene The latter can be dispensed with if said expressible DNA 25 sequence which is ?anked by homologous DNA sequences sequence itself already has su?iciently great homology with at a site. which is de?ned on the basis of the homologies corresponding DNA regions within the bacteria/genome so present between inserted DNA and DNA intrinsic to the that direct exchange of this DNA sequence for said homolo bacteria. within the bacterial genome by homologous recom gous genomic DNA can take place by means of homologous bination. The ?anking. homologous DNA sections are. recombination. moreover. within the scope of this invention present. pref Besides double-stranded DNA. it is also possible to erably fused together to a unit. as component of a DNA employ in the process according to the invention single molecule which is closed in the form of a ring. stranded DNA and partially single-stranded DNA. In a preferred embodiment. moreover. the DNA which is Suitable for use in the process according to the invention to be inserted is integrated into a plasmid which either are both homologous and heterologous gene(s) or DNA 35 already contains homologous DNA sections or else acquires sequences. as Well as synthetic gene(s) or DNA sequences the latter cloned in at a later time. complying with the de?nition made within the scope of the Thus. if the intention is to integrate not just single genes present invention. or gene fragments but the complete plasmid DNA. which The DNA sequences which are to be integrated can. may contain said genes or gene fragments. into the myxo moreover. be constructed exclusively from genomic. from bacterial genome. it is sufficient to clone said homologous cDNA or synthetic DNA. Another possibility comprises the DNA sequences into the plasmid DNA at a required site. construction of hybrid DNA sequences consisting both of although. where possible. the genes intended for expression cDNA and of genomic DNA and/or synthetic DNA. should be functionally retained. Thus. it is also possible in In this case. the cDNA can originate from the same gene this case too for the DNA which is to be integrated (plasmid or DNA section as the genomic DNA or else both the cDNA 45 DNA) to be regarded in principle as ?anked by homologous and the genomic DNA can originate from different genes or DNA sequences because these homologous DNA sections DNA sections. In each case. however. it is possible for both can be thought of as fused together to a unit within the DNA the genomic DNA and/or the cDNA. each on its own. to be molecule which is closed in the form of a ring. prepared from the same or from different genes or DNA Besides structural genes. it is also possible to use any sections. 50 other desirable genes or gene fragments or other useful DNA If the DNA sequence contains portions of more than one sequences such as. for example. binding sites of regulator gene or DNA section. these can derive either from one and molecules. promoters. terminator sequences etc. the same organism. from a plurality of organisms. which It is now possible for the ?rst time. by the choice of belong to various strains. or varieties of the same species or suitable DNA sequences of homologous or heterologous different species of the same genus. or else from organisms 55 origin. which have sufficiently great homologies with cor which belong to more than one genus thereof or to another responding sections within the bacterial genome and thus taxonomic unit. allow exchange of genetic material via homologous In order to ensure the expression of a structural gene in the recombination. for genes or other DNA sequences to be bacterial cell. it is possible. where appropriate. for the integrated speci?cally at predetermined sites in the myxo coding gene sequences initially to be linked in an operable bacterial genome and to be expressed there where appropri manner to expression sequences able to function in the ate. myxobacteria] cell. The extent of the homology. which is necessary for The expressible hybrid gene constructions of the present exchange via homologous recombination. between the invention thus usually contain. besides the su'uctural gene homologous DNA sections and the corresponding genomic (s). also expression signals which include both promoter and 65 DNA region depends on a variety of parameters and must terminator sequences and. preferably. further regulatory therefore be adapted to the appropriate needs in each case. sequences of the 3' and 5‘ non-translated regions. depending on the DNA sequences used. It is assumed on the 5,686,295 10 present state of knowledge that a homologous region com clone them into a suitable plasmid. The ligation of homolo prising at least 100 Bp is su?icient to bring about the gous DNA fragments and of DNA fragments of homologous required recombination event. and heterologous origin into a suitable cloning vector is A homologous region which extends over a range of 0.3 carried out using standard methods as are described. for to 4 Kb. but preferably over a range of l to 3 Kb. is preferred example. by Maniatis et al, 1982. within the scope of this invention. This usually entails the vector and the DNA sequence Suitable for use as homologous DNA sections within the which is to be integrated initially being cut with suitable scope of this invention are primarily DNA sequences of restriction enzymes. Examples of suitable restriction homologous origin which can be obtained by isolation of the enzymes are those which provide fragments with blunt ends. complete myxobacterial DNA and subsequent digestion 10 such as. for example. Smal. HpaI and EcoRV. or else with suitable restriction enzymes. Where the DNA sequence enzymes which form cohesive ends. such as. for example. of said homologous DNA fragments is known they can. of EcoRL SacI. BamHI. Sall. PvuI etc. course. also be prepared by synthesis. Both fragments with blunt ends and those with cohesive However. it is furthermore also possible to use homolc» ends. which are complementary with one another. can be gous DNA sections of heterologous origin. which have been 15 linked again. with the aid of suitable DNA ligases. to give a isolated not directly from the genome of the target organism single continuous DNA molecule. but. for example, from related organisms and which thus do Blunt ends can also be prepared by treatment of DNA not necessarily have 100% identity with the corresponding fragments which have protruding cohesive ends with the DNA regions on the genome of the target organism but are Klenow fragment of E. coli DNA polymerase by ?lling in only essentially homologous with the latter. that is to say the gaps with the appropriate complementary nucleotides. have a degree of homology between 60% and 100%. pref On the other hand. cohesive ends can also be prepared erably between 80% and 100% and very particularly pref arti?cially. for example by attaching complementary erably between 90% and 100%. homopolymeric tails to the ends of a required DNA An essential component of the present invention is there sequence and of the cut vector molecule using a terminal fore formed by a process for the speci?c genetic manipula 25 deoxynucleotidyl transferase or else by attaching synthetic tion of bacteria of the order Myxobacterales. which is oligonucleotide sequences (linkers) which carry a restriction characterised in that genetic material of homologous origin cleavage site. and subsequent cutting with the appropriate or a combination of genetic material of homologous and enzyme. heterologous origin is inserted into the myxobacterial cell It is possible in principle to use for the preparation and and integrated there via homologous recombination speci? 30 multiplication of the constructs which have been described cally at a site. which is accurately de?ned by reason of the above and which contain DNA fragments of homologous or homologies present. into the chromosome of said myxobac else a combination of DNA fragments of homologous and teria. heterologous origin all conventional cloning vectors such as. It is thus now possible for the ?rst time within the scope for example. plasmid or bacterophage vectors as long as they of this invention. by preparing appropriate hybrid gene 35 have replication and control sequences which originate from constructions in the manner described above. to carry out species which are compatible with the host cell. speci?c modi?cations of bacteria-intrinsic genes within the The cloning vector usually carries an origin of replication. myxobacterial genome or else to incorporate additional in addition speci?c genes which lead to phenotypical selec genes or other DNA fragments into the myxobacterial tion features in the transformed host cell. especially to genome. If the integration takes place within a functional resistance against antibiotics. The transformed vectors can gene or operon. this usually leads to inactivation thereof and. be selected on the basis of these phenotypical markers after as a consequence. to a phenotypically observable defect. a transformation into a host cell. The speci?c procedure for this can be such that myxo Selectable phenotypical markers which can be used bacterial cells are transformed with one of the recombinant within the scope of this invention comprise. for example. DNA molecules described above. with the genes. hybrid 45 without this representing a limitation on the subject-matter gene constructions or other DNA fragments contained in of the invention. resistances to ampicillin. tetracycline. said recombinant DNA molecule being integrated by chloramphenicol. hygromycin. G418. kanamycin. neomycin homologous recombination randomly or else. preferably. and bleomycin. A prototrophy for particular amino acids can speci?cally at a site. which is defined by reason of the function as further selectable marker. for example. homologies present and thus can be predetermined. into the 50 Preferred within the scope of the present invention are bacterial genome. primarily E. coli plasmids such as. for example. the plasmid In a speci?c embodiment of the present invention. the pSUP2021 used within the scope of the present invention. insertion of the genetic material into myxobacteria. in par Suitable host cells for the cloning described above which ticular into myxobacteria of the Sorangium/Polyangium are Within the scope of this invention are primarily group. takes place in an undirected manner via conjugal 55 prokaryotes. including bacterial hosts such as. for example. transfer of plasmid DNA from a donor cell to a Sorangium/ AJumefaciens, E. coli, S. typhimurium and Serratia Polyangium recipient cell. marcescens, furthermore pseudomonads. actinomycetes. The procedure for the preparation of suitable plasmids salrnonellae and myxobacteria themselves. which have a homology with the myxobacterial chromo E. coli hosts such as. for example. the E. coli swain some which is su?icient for integration via homologous HCBlOl are particularly preferred. recombination can be. for example. such that the complete Competent cells of the E. coli strain H3101 are in this DNA is initially isolated from myxobacteria and subse connection prepared with the aid of the processes custom quently fragmented. This fragmentation can be carded out arily used for the transformation of E. coli [see: “General either mechanically by the action of shear forces or else. recombinant DNA techniques"]. preferably. by using suitable restriction enzymes. 65 Transformation and subsequent isolation on a suitable It is then possible to isolate from the large number of medium are followed by the resulting colonies being sub resulting fragments those of suitable size and subsequently jected to dilferential screening by plating out on selective 5,686,295 11 12 media. It is then subsequently possible to isolate the appro example. pSUPlOl, pSUP30l. pSUP401, pSUP20l. priate plasmid DNA from those colonies which contain pSUP202. pSUP203 or pSUP205. and the derivatives plasmids with DNA fragments cloned in. derived therefrom [Simon et al (1988)] can likewise be used Recombinant plasmids of different size are obtained in within the scope of the process according to the invention. this way. After restriction analysis it is then possible for During the course of the experiments carried out within plasmids of suitable size to be selected for the subsequent the scope of this invention it has emerged that it is advan insertion of the plasmid DNA into the myxobacterial cell. tageous when the myxobacterial recipient is exposed to a This DNA transfer can moreover take place either directly or brief heat treatment during the course of the conjugal else. preferably, via an intermediate host (donor cell) within transfer before the incubation with the donor strain. A the scope of a conjugal transfer. 10 preincubation of the recipient cell at a temperature of 35° C. An essential component of the present invention therefore to 60° C., preferably at a temperature of 42° C. to 55° C. and relates to the construction of plasmids which, besides very particularly preferably at a temperature of 48° C. to 52° homologous sections. can also contain one or more gene C. for one to 120 minutes. but in particular 5 to 20 minutes. constructions consisting of one or more structural genes or is preferred. other desirable genes or gene fragments which are. where Used in a prefm'red embodiment of the present invention appropriate. linked in an operable manner to expression is an E. coli donor strain which contains the transfer genes signals able to function in bacterial cells. or other useful (tra) of plasmid RP4 incorporated into the chromosomal DNA sequences. The homologous DNA fragments can in DNA. Preferred within the scope of this invention is the E. this connection either consist entirely of genome sections coli donor strain W3 101(pME30S) which contains the which are intrinsic to the bacteria (myxobacteria) and thus helper plasmid pME3 05 which has the transfer function (Ira) are completely of homologous or else they can. of RP4. besides homologous sections. also contain more or less Particularly interesting for the process technique. and thus expressed portions of heterologous origin. The use of particularly preferred within the scope of this invention. are homologous DNA sections of purely heterologous origin is bacterial strains which are suitable both as hosts for cloning also conceivable. 25 of vectors with integrated DNA sequences and for use as These plasmids can be used in a further process step for donor cell within the scope of the conjugal transfer. Like insertion of the genetic constructions which have been wise particularly preferred are bacterial strains which are described above and which contain. where appropriate, a restriction-negative and thus do not degrade inserted foreign structural gene which codes for a required gene product. into DNA. Both of the abovementioned criteria are met by the E. the myxobacterial cell and integration there into the bacterial 30 coli strain ED8767(pUZ8) in an ideal manner. but this is genome. mentioned at this point only as representative of other The transfer of the genetic constructions according to the suitable bacterial strains and is not intended to limit the application into the myxobacterial cells can be carded out in application in any way. a variety of ways. Preferred within the scope of this inven Besides the conjugal gene transfer. described above. from tion is conjugal transfer from a donor cell to the myxobac 35 a donor cell into a myxobacterial recipient. it is. of course. terial recipient. also possible to use other suitable gene transfer processes for It is possible within the scope of this conjugal transfer for inserting genetic material into myxobacteria. Mention may the DNA which is to be transferred moreover to be either be made here primarily of gene transfer via electroporation. initially cloned. as described above. in one of the cloning within the scope of which the myxobacterial cells are brie?y vectors customarily used. and subsequently transformed into exposed to high electric ?eld strengths [Kuspa and Kaiser a suitable intermediate host which functions as donor cell. (1989)]. The general outline conditions for electroporation The roundabout route via the intermediate host can be of prokaryotic cells are described in detail in US. Pat. No. avoided by using a host strain which is suitable both for the 4.910.140. cloning of DNA and for the use as donor cell within the NON-LIMITING EXEMPLARY EMBODIMENTS scope of the conjugation. Intermediate hosts which can be used within the scope of General Recombinant DNA Techniques this invention as donor cells are essentially prokaryotic cells Since many of the recombinant DNA techniques used in selected from the group consisting of E. coli, this invention are routine for the person skilled in the art. a pseudomonads. actinomycetes. salmonellae and myxobac brief desuiption of these generally used techniques is to be 50 teria themselves. given below. All these processes are described in the refer The precondition for conjugal transfer of plasmid DNA ence of Maniatis et al (1982). unless separate reference is from a donor cell to a recipient is the presence of transfer made thereto. (Ira) and mobilisation functions (mob). Moreover the mobilisation function must contain at least the transfer A. Cutting with restriction endonucleases origin (oriT) and be located on the plasmid to be transferred SS Typically. the reaction mixture contains about 50 to 500 By contrast. the transfer function (tra) can be either located ug/ml DNA in the buffer solution recommended by the on the plasmid or on a helper plasmid or else be present manufacturer. primarily New England Biolabs. Beverly. integrated into the chromosome of the donor cell. Mass. and Bohringer. Mannheim (FRG). 2 to 5 units of Plasmids which meet the abovementioned precondition restriction endonucleases are added for each pg of DNA and and are therefore preferred within the scope of this invention the reaction mixture is incubated at the temperature recom essentially fall into incompatibility groups P. Q. T, N. W and mended by the manufacturer for one to three hours. The C011. The prototype of the P group plasmids is the plasmid reaction is stopped by heating at 65° C. for 10 minutes or by RP4. Particularly preferred within the scope of this inven extraction with phenol. followed by precipitation of the tion is the plasmid pSUP2021 which contains a 1.9 Kb DNA with ethanol. This technique is also described on pages fragment from the plasmid RP4. which has as component of 65 104 to 106 of the Maniatis et a1 (1982) reference. the mob function (RP4mob) the n'ansfer origin (oriT). Other B. Treatment of the DNA with polymerase in order to plasmids with the mob function (RP4mob). such as. for generate blunt ends. 5,686,295 13 14 50 to 500 pg/ml DNA fragments are added to a reaction restriction endonuclease. one unit of alkaline phosphatase mixture in the buffer recommended by the manufacturer, from the intestine of calves. which has been obtained from primarily New England Biolabs. Beverly, Mass. and B Boehn'nger-Mannheim. Mannheim. is added. The DNA is ohringer. Mannheim (FRG). The reaction mixture contains incubated at 37° C. for one hour and subsequently twice all four deoxynucleotide triphosphates in concentrations of extracted with phenol and precipitated with ethanol. 0.2 mM. The reaction is carried out at 15° C. for 30 minutes F. Linkage of the DNA ?agments and is then stopped by heating at 65° C. for 10 minutes. For When fragments with complementary cohesive ends are fragments which are obtained by cutting with restriction to be linked together. about 100 ng of each fragment are endonucleases which generate 5'-protruding ends. such as incubated in a reaction mixture of 20 to 40 pl with about 0.2 EcoRI and BamHI. the large fragment. or Klenow fragment. 10 units of T4 DNA ligase from New England Biolabs in the of DNA polymerase is used. For fragments which are bu?’er recommended by the manufacturer. The incubation is obtained by endonucleases which generate 3‘-protruding carried out at 15° C. for 1 to 20 hours. When DNA fragments ends. such as PstI and Sand. T4 DNA polymerase is used. with blunt ends are to be linked. they are incubated as above The use of these two enzymes is described on pages 113 to apart from the amount of T4 DNA ligase being increased to 121 of the Maniatis et al ( 1982) reference. 2 to 4 units. C. Agarose gel electrophoresis and puri?cation of DNA fragments from gels G. Transformation of DNA into E. coli The agarose gel electrophoresis is carded out in a hori The E. coli strains H8101. W3101 and ED8767 are used for most of the experiments. DNA is introduced into E. coli zontal apparatus as described on pages 150 to 163 of the by the calcium chloride process as has been described by Maniatis et a1. reference. The bu?er used is the his-acetate 20 bu?’er described therein. The DNA fragments are stained by Maniatis et al (1982). pages 250 to 251. 0.5 pg/mi ethidium bromide which is either present in the gel H. Screening of E. coli for plasmids or tank butfer during the electrophoresis or added after the After the transformation. the resulting colonies of E. coli electrophoresis. The DNA is visualised by illumination with are tested for the presence of the required plasmid by a rapid long-wavelength ultraviolet light. 25 plasmid isolation process. Two usual processes are described When the fragments are to be removed from the gel. the on pages 366 to 369 of the Maniatis et al (1982) reference. agarose used is one which gels at low temperature and can 1. Isolation of plasmid DNA on a large scale be obtained from Sigma Chemical. St. Louis. Miss. After the Processes for the isolation of plasmids from E. coli on a electrophoresis. the required fragment is cut out. placed in a 30 large scale are described on pages 88 to 94 of the Maniatis plastic tube. heated at 65° C. for about 15 minutes. extracted et a1 (1982) reference. three times with phenol and precipitated twice with ethanol. This process is a slight modi?cation of that described by EXAMPLES Maniatis et al (1982) on page 170. As alternative. the DNA can be isolated from the agarose Example 1 with the aid of the Geneclean kit (Bio 101 Inc.. La Jolla. 35 Calif.. USA). Cultivation conditions for Sorangium D. Addition of synthetic linker fragments onto DNA ends Sorangium cellulasum is cultivated in a G51b Liquid Ifit is required to attach a new endonuclease cleavage site medium [see section “Media and buffers] at a temperature of onto the end of a DNA molecule. the molecule is. where 30° C. The cultures are aerated by shaking at 180 rpm. It is appropriate. initially treated with DNA polymerase in order also possible to use a G52c medium as alternative medium. to generate blunt ends as described in the above section. The Sole medium described in the section “Media and About 0.1 to 1.0 pg of the fragment is added to about 10 ng bu?ters” can be used for the cultivation on solid medium. of phosphorylated linker DNA. which has been obtained The incubation temperature is 30° C. in this case too. from New England Biolabs. in a volume of 20 to 30 pl with 2 g1 of T4 DNA ligase from New England Biolabs. and 1 Example 2 mM ATP in the buffer recommended by the manufacturer. After incubation at 15° C. overnight. the reaction is Cultivation conditions for E. coli stopped by heating at 65° C. for 10 minutes. The reaction E. coli cells are cultivated in an LB medium [Miller mixture is diluted to about 100 pl in a buffer which is correct 50 for the restriction endonuclease which cuts the synthetic (1972)] at a temperature of 37° C. linker sequence. Approximately 50 to 200 units of this endonuclease are added. The mixture is incubated at the Example 3 appropriate temperature for 2 to 6 hours. and then the Preparation of a streptomycin-resistant spontaneous fragment is subjected to an agarose gel electrophoresis and 55 puri?ed as described above. The resulting fragment will now mutant of Sorangium cellulosum have ends with endings which have been generated by 200 pl of a three-day old Sorangium cellulosum culture cutting with the restriction endonuclease. These ends are [wild-type strain So oe 26] which has been raised in liquid usually cohesive so that the resulting fragment can now medium is plated out on solid medium [Sole medium] which easily be linked to other fragments with the same cohesive is supplemented with 300 pgml streptomycin. The incuba ends. tion time is 14 days at a temperature of 30° C. The colonies E. Removal of 5'-terminal phosphates from DNA frag growing on this medium are spontaneous streptomycin ments resistant mutants which are cultivated once more on the During the plasmid cloning steps. treatment of the vector same medium (with streptomycin) for further concentration plasmid with phosphatase reduces the recircularisation of 65 and puri?cation. the vector (discussed on page 13 of the Maniatis et al One of these streptomycin-resistant colonies is selected reference). After the DNA has been cut with the correct and is called S J3. A sample of this mutated Sorangium 5 ,686,295 15 16 cellulosum So ce 26 strain was deposited on 25.01.1991 at subjected to a differential screening by parallel plating out the "Deutsche Sammlung von Mikroorganismen und on ampicillin-containing [60 pg/ml] and ampicillin-free Zellkulturen GmbH” [Braunschweig, FRG]. which is rec medium. It is subsequently possible to isolate those colonies ognised in accordance with the provisions of the Budapest which have lost their ampicillin resistance due to the inte Treaty as international depository, under deposit number gration of the Sorganium (sic) DNA fragments. The plas DSM 6380. mids are then isolated from these ampicillin-sensitive colo mes. Example 4 Recombinant plasmids of different size are obtained in this way. After restriction analysis. three of these plasmids Preparation of the complete DNA of Sorangium are selected for further experiments. These plasmids. called To isolate the complete DNA. a Sorangium culture in the pSlBSO. pSIB55 and pSJB58, contain Sorangium DNA stationary phase is centrifuged at 10.000 rpm for 10 minutes. inserts of 1 Kb. 3.5 Kb and 4 Kb respectively. The cells are removed and resuspended in STE butfer [see 5.1.2. Conjugative transfer of plasmid pSJB55 into Sor section “Media and buifers"] and adjusted to a cell density angium cellulosum of about 109 cells/ml. The transfer of plasmid pSJB55 into Sorangium cellulo Subsequently 450 pl of this suspension are mixed with sum takes place with the mediation of E. coli strain W310i 200 pl of RLM buffer [see section “Media and buffers”] and (pME305) [Jaoua Set al (1987)]. which is capable of a 2 pl of diethyl pyrocarbonate. Thorough and uniform mixing conjugation-like information exchange with Sorangium. The are ensured by using suitable equipment such as. for E. coli plasmid pME305 [Rella (1984)] is in this case used example. a Vortex or the like. After incubation in an incu as helper plasmid for the mobilisation of pSIBSS. bator at 70° C. for 30 minutes. 100 pl of potassium acetate Initially. competent cells of the E. coli strain W3101 [5 M] are added. This mixture is incubated on ice for 15 (pME305) are transformed. with the aid of the processes minutes and thoroughly mixed [Vortex] every 5 minutes. normally used for the transformation of E. colt‘, with 5 pl of Alter centrifugation [15 minutes at 10.000 rpm] the super 25 the previously isolated pSJBSS plasmid DNA. The trans natant is subsequently mixed with 500 pl of phenol! formed E. coli cells are thereby becoming the donor for the chloroform/isoamyl alcohol [25/24/1] for extraction of the plasmid pSJB55. (sic) proteins. After renewed centrifugation [15 minutes at 10.000 rpm] the upper phase which contains the DNA fraction is For the actual transfer. 15 ml of a Sorangium cellulosum removed. and any phenol content still present therein is S13 culture [4x108 cells/ml to 1-4><109 cells/ml] in the removed with diethyl ether [1 ml]. The DNA is then pre 30 stationary phase are mixed with 10 ml of a late log phase cipitated by adding 1 mi of ethanol. After incubation at -70° culture of E. coli donor cells which contain a comparable C. for 30 minutes. the complete mixture is centrifuged at content of cells. These are then centrifuged together at 4000 10.000 rpm for 15 minutes. the pellet is washed with 70% to 8000 rpm for 10 minutes and resuspended in 500 pl of a ethanol and dried in vacuo. Finally. the DNA is dissolved in G51b or G511 medium. TER buifer. 35 It has proved advantageous for the Sorangium recipient cells to be exposed brie?y to a heat treatment in a waterbath Example 5 before the conjugation with E. coli. The best transfer results with the Sorangium cellulosum strain S 13 can be achieved Conjugative transfer of pSJBSS into Sorangium with a heat treatment at a temperature of 50° C. for 10 cellulosum S J3 minutes. Under these conditions transfer frequencies of 5.1 Two-stage process 1-5xl0‘5 can be achieved. which corresponds to an increase 5.1.1 Cloning of Sorangium DNA into plasmid by a factor of 10 compared with a process without previous pSU'P2021 heat treatment. The chromosome isolated from the Sorangium S 13 strain Transfer to plates with Sole solid medium is followed by is cut with the restriction enzyme PvuI. The fragments a incubation at 30° C. for two days. The cells are then obtainable in this way are cloned into the plasmid harvested and resuspended in 1 ml of G51b or G511 pSUP202l [Simon R. et al (1983]). This entails 0.2 pg of medium. 100 pl of this bacterial suspension are plated out on plasmid DNA and 1 pg of chromosomal DNA being initially a selective Sole medium which. besides kanamycin [25 digested with PvuI and subsequently precipitated with etha mg/l] also contains phleomycin [20 to 35 mg/l] and strep nol. The precipitate is removed. dried and the dried pellet is tomycin [300 mg/l] as selective agents. Counter-selection of suspended in 14 pl of double-distilled water. Then 2.5 pl of the donor swain [E. colt‘ W3101 (pME305)] is carried out a ten-fold concentrated ligation buffer [see section “Media with the aid of streptomycin. and bu?ers"]. 2.5 pl of bovine serum albumin [0.1%]. 2.5 pl The colonies growing on this selective Sole medium after an incubation time of 10 to 14 days are transconjugants of ofATP [10 mM]. 2.5 pl of D'I'I‘ [0.2M]. 8 p1 of H20 and 1 55 pl of T4 DNA ligase are added. The complete ligation Sorangium cellulosum which have acquired phleornycin mixture is then incubated at a temperature of about 8° C. resistance owing to conjugative transfer of the plasmid overnight. pSJBSS. These phleomycin-resistant colonies can be used 5 pl of this ligation mixture are transfcn'med into the E. for the subsequent molecular biological investigations. The coli strain HB101 for the cloning of recombinant plasmids. transformation frequency for the transfer of plasmid pSJBSS For this. competent cells of the E. coli strain HB101 are to Sorangium averages 3X10_6 based on the recipient strain prepared with the aid of the processes normally used for the SJ3. transformation of E. coli [seer “General recombinant DNA The plasmids P813 50 and pSJB58 can be transferred to techniques”]. Sorangium in an analogous manner. After transformation and subsequent incubation for 24 5.2 One-stage process hours on LB agar supplemented with kanamycin [25 pg/ml] 5.2.1 Cloning of Sorangium DNA into plasmid and chloramphcnicol [25 pg/ml]. the resulting colonies are pSUP202l 5,686,295 17 18 The cloning of Sorangium DNA into plasmid pSUPZOQl The complete DNA which has been isolated from the can be carried out as described in Example 5.1.1. transformed Sorangium cells as described above [compare Owing to the helper plasmid pME305 used in 5.1.1 being Example 4] is digested with SmI and SalI and loaded onto exchanged for the plasmid pUZ8 [Hedges and Matthew a horizontal tris-acetate [40 mM tris-HCl. 20 mM sodium (1979)] which. in contrast to the abovementioned plasmid. acetate. 2 mM EDTANaZ. pH 7.8] agarose gel [0.9%]. After carries no ampicillin-resistance gene. the cloning step in the the electrophoresis the gel is placed initially in a denaturing E. coli intermediate host H3101 can be dispensed with solution [1.5M NaCl. 0.5M NaOH] for 30 minutes and because direct cloning in the E. coli donor strain ED8767 subsequently in a neutralising solution [1.5M NaCl. 0.5 M which is intended for the conjugal transfer is now possible. tris-HCl. 1 mM EDTANa2. pH 7.2] . The DNA is transferred. The plasmid pUZ8 is a derivative of the plasmid RP4 10 by means of a Southern capillary blotting. using a 20-fold which covers a wide host range and is described by Datta et concentrated SSC buffer [see section "Media and bu?ers”] al (1971). The modi?cations compared with the initial onto a nylon membrane [for example an Amersham Hybond plasmid RP4 relate essentially to the ampicillin-resistance nylon membrane; Amersham International plc. Amersham gene and to the insertion element IS21. both of which are Place. Amersharn. England HP7 9NA] and ?xed there by deleted. and to the incorporation of an additional gene which 15 UV treatment for 6 minutes. Further details of this process confers resistance to mercury ions [see Jaoua et a1 (1987)]. are described in the Amersham International handbook “Membrane Transfer and Detection Methods”. (1985). The ligation mixture prepared as in Example 5.1.1 can therefore now be transformed directly into the E. coli strain The DNA intended as hybridisation probe is labelled by HD8767. For this. competent cells of the E. coli strain means of a nick translation [Rigby D. W. J. et al (1977)]. 20 ED8767 are prepared with the aid of the processes custom This takes the form of the 32P-labelled PvuI insert compris arily used for the transformation of E. coli [seez “General ing 3.5 Kb from the plasmid pSJB55. The actual hybridi recombinant DNA techniques"]. sation is carried out using a slight modi?cation of the process of Denhardt [Denhardt DT (1976)]. The buffer used After transformation and subsequent incubation on LB for the prehybridisation and hybridisation has the following agar supplemented with tetracycline [10 pg/ml] and 25 chloramphenicol [25 pg/mi] for 24 hours. the resulting composition: 6>

Glucose 0.2% Bacterial strains and plasmids Starch 0.5% [potato starch, Noredux type; CERESTAR ITALIA Relevant characteristics S.p.a., Milan, Italy] Strain Tryptone [MARCQ Hackensack. NJ USA] 0.2% 25 Probion S 0.1% Escherichia coli [Single Cell Protein; HO'CHST AG. Frankfurt, FRG] CaCl; X 2 E20 0.05% W3101Na1 RecAlB, trpE, NalR mgso, x 7 E20 0.05% E8101 F- , hsds20 (r-, 111-), recA13, am 14, I-IEPES [I‘IUKA] 1.2% proAZ, 1mm, galKZ, rpsmo (SmR), G520 medium (pH 7.4) xyl-S, mtl-l, sup E 44, lambda 30 ED8767 recA, supE, supF. hsdS Glucose 2.0 gll M Starch 8.0 gll [potato starch, Noredtnr type; CERESTAR ITALIA So ce 26 Wild-type strain S.p.a., Milan, Italy] So ce 26/513 SmR spontznmus mutant Plasmid Soya meal defatted 2.0 II 35 [MUCEDOLA S.r.l., Settimo Milanese, Italy] pSUP2021 Ap. Cm, Km, Ph Yeast extract 2.0 g/l psmso Cm, Km, Ph [FOULD & SPRINGER1 Maison Alfort1 France] pSJ'BSS Cm, Km, Ph c11012 >< 2 H20 1.0 g/l pSIB58 Cm, Km, Ph MgSO,‘ x 7 11,0 1.0 g/l pME305 Ap, Tc Fe-EDTA [8 g/l stock solutirm] 1.0 m1 pUZ8 Tc, Km, Hg HEPIE [FLUKA] 2.0 311 Distilled water ad 1000 m1 pH is adjusted to 7.4 with NaOH before sterilisation [20 minutes at 120° C.]. pH after sterilisation: 7.4 DEPOSITION 501E medium (pH 7.4) Within the scope of the present application. the following 45 Glucose‘ 0.35% microorganisms and plasmids have been deposited at the 'Iryptone [MARCO, Hackensack, NJ USA] 0.05% "Dcutsche Sammlung von Mikroorganismen und Zellkul MgSO‘ X 7 1120 0.15% turen GmbH" in Braunschweig (FRG). which is recognised Ammonium sulfate‘ 0.05% in accordance with the Budapest Treaty as international ctcl2 x 2 1120* 0.1% depository. to comply with the requirements for the inter 112mm; 0.006% 50 Sodium dithionite" 0.01% national recognition of the deposit of microorganisms for the Fe-EDTA' 0.000896 purpose of patenting. HEPES lFLUKA] 1.2% Supematant of a sterilised. 3.5% (v/v) stationary S. cellulosum culture" Microorganism! Date of Deposit Date of viability Agar 1.5% 55 plasmid deposit number certi?cate ‘Addition takes place only after sterilisation pH is adjusted to 7.4 with NaOH before sterilisation pSJB55 25.01.1991 DSM 6321 25.01.1991 [20 minutes at 120° C.]. (cloned into E. 0011) LB medium Somngium cellulosunl 25.01.1991 DSM 6380 14.02.1991 So ce 26/813 'll'yptone 10.0 g]! Yeast extract 5.0 g1 NaCl 5.0 g/l LIST OF REFERENCES STE bu?er (pH 8.0) Breton A. M. et al. J Bacteriol. 161:523-528 (1985) Sucrose 25% EDTANa2 1 mM 65 Breton A. M. et al. J Biotechnol. 4:303-311 (1986) 'l'tis-HCl 10 mM Breton A. M. and Guespin-Michel J. F.. FEMS Microbiol Lett. 4-—0:183—188 (1987) 5,686,295 21 22 Datra N. et al, J Bacteriol 108:1244-1249 (1971) wherein said ?rst or second DNA sequence is obtained by Denhardt D. T.. Biochem Biophys Res Commun. fragmentation of the Sorangium cellulosum genome, 23:641-646 (1976) and further wherein the homology is such that upon transformation of Sorangium cellulosum with the Hedges R. W. and Matthew M.. Plasmid 2:269-278 recombinant DNA molecule. integration of said ?rst or (1979) second DNA sequence by homologous recombination Gentz R. et al. Proc Natl Acad Sci. USA 7—8:4926-4940 occurs at a site de?ned by the homology between the (1981) integrated DNA and the Sorangium cellulosum Jaoua S. et a1. Plasmid 18: 111-119 (1987) genomic DNA, independent of structural elements present on the Sorangium cellulosum chromosome. Jaoua S. et a1. Plasmid 23: 183-193 (1990) 10 wherein said integration is not lethal to Sarangium Kaiser D., Genetics of Myxobacteria. in: "Myxobacteria: cellulosum. Development and Cell Interactions”, ed E. Rosenberg, pp 2. The recombinant DNA molecule of claim 1. wherein 163-184. Springer Verlag, Berlin/New York (1984); said homologous integrated DNA sequence is homologous Kuner Y. M. and Kaiser D.. Proc Natl Acad Sci USA. with corresponding DNA regions in the Somngium cellulo 78:425-429 (1981) sum genome. Kuspa A. and Kaiser D.. J Bacteriol 17122762-2772 3. The recombinant DNA molecule of claim 1. wherein said integrated DNA sequence is double-stranded. (1989) 4. The recombinant DNA molecule of claim 1. wherein Maniatis T. et al. “Molecular Cloning”. Cold Spring said integrated DNA sequence is operably linked to an Harbor Laboratory. Cold Spring Harbor, N.Y. (1982) 20 expression sequence functional in Sorangium cellulosum. Miller J. H.. "Experiments in Molecular Genetics". Cold 5. The recombinant DNA molecule of claim 1. wherein Spring Harbor Laboratory. Cold Spring Harbor. N.Y. (1972) said integrated DNA sequence is ?anked by a DNA Murray N. E. et al. Mol Gen Genet 150:53 (1977) sequence homologous to a corresponding DNA region Within the Sarangium cellulosum genome. O’Conner K. A. and Zusman D. R.. J Bacteriol. 6. The recombinant DNA molecule of claim 1. wherein l55:317-329 (1983) said integrated DNA sequence originates from the target Rella M.. Dissertation ETH Zurich. No. 7601. SFITZ Sarangium cellulosum genome. Reichenbach H. et al. Trends in Biotechnology. 7. A cloning vector comprising the recombinant DNA 6:115-121 (1988) molecule of claim 1. Rigby D. W. J. et al. J Mol Biol. 113:237-251 (1977) 30 8. A plasmid comprising the recombinant DNA molecule of claim 1 and a mobilization function active in Sorangium Rosenberg M. and Court D.. Ann Rev Genetics cellulosum. 13:319-353 (1979) 9. A genetically modi?ed Sorangium cellulosum cell Shimkets L. J. et al. Proc Natl Acad Sci USA. comprising the recombinant DNA molecule of claim 1. 80:1406-1410 (1983) 35 10. The recombinant DNA molecule of claim 5. wherein Simon R. et a1. Bio/Technol. November 1983:784-791 said ?anking DNA sequences are linked to form a single (1983) continuous DNA molecule. EP 0 310 619 11. The genetically modified Sorangium cellulosum cell EP 0 358 606 of claim 9. wherein said recombinant DNA molecule is integrated into the cell’s genome via homologous recombi US. Pat. No. 4.910.140 nation at a site de?ned by the homology between the DNA We claim: sequence and the corresponding regions in the genome. 1. A recombinant DNA molecule that integrates into the independent of structural elements present in the genome. or genome of Sorangium cellulosum, comprising: of specific transposition events. a ?rst DNA sequence homologous with corresponding 12. A genetically modi?ed Sarangium cellulosum cell DNA regions in the Sorangium cellulosum genome. or comprising an exogenous DNA sequence integrated into its a second DNA sequence ?anked by ?anking DNA genome via homologous recombination. sequences homologous with corresponding DNA regions in the Sarangium cellulasum genome. * * * * *