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\Iiiiiiiiiiiiiillilllllllllillll||L||L||Llllllilllllllllllllllllllllllllius005686295a United States Patent [191 [11] Patent Number: 5,686,295 Jaoua Et Al

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\Iiiiiiiiiiiiiillilllllllllillll||L||L||Llllllilllllllllllllllllllllllllius005686295a United States Patent [191 [11] Patent Number: 5,686,295 Jaoua Et Al \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
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