Abstract:
There is provided DNA sequences isolated from  Myxococcus xanthus  partially encoding a functional portion of a polypeptide component required for the synthesis of antibiotic TA. Also provided are purified, isolated and cloned DNA sequences encoding a polypeptide component required for postmodification of antibiotic TA and encoding a gene product involved in the regulation of the biosynthesis of antibiotic TA. A purified, isolated and cloned DNA sequence having a DNA sequence (seq. ID No:2 and 20) encoding a polypeptide component required for encoding the TA gene cluster and any mutations thereof is provided. Also provided are methods of using the TA genes for combinatorial genetics and of using the TA genes encoding for synthesis and modification or regulation of antibiotic TA.

Description:
CROSS REFERENCES TO RELATED APPLICATIONS 
     This is a continuation-in-part of U.S. patent application Ser. No. 09/240,537, filed Jan. 29, 1999, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     Polyketides constitute a large and highly diverse group of secondary metabolites synthesized by bacteria, fungi and plants, with a broad range of biological activities and medical applications. They include anti-cancer agents (Daunorubicin), antibiotics (tetracyclines, erythromycin etc.), immunosuppressants (macrolide FK506) and compounds with mycotoxic activity (aflatoxins, ochratoxins, ergochromes, patulin etc.). Polyketides are synthesized by repetitive condensations of acetate or propionate monomers in a similar way to that of fatty acid biosynthesis. Structural diversity of polyketides is achieved through different thioester primers, varying chain extension units used by the polyketide synthases (PKSs), and variations in the stereochemistry and the degree of reduction of intermediates. Diversity is also achieved by subsequent processing, such as alkylations, oxidations, O-methylations, glycosylations and cyclizations. Genetic studies indicated that gene organization of functional units and motif patterns of various PKSs are similar. This similarity was used to identify and obtain new PKS systems in both gram negative and gram positive bacteria. 
     PKS systems are classified into two types: type I PKSs are large, multifunctional enzymes, containing a separate site for each condensation or modification step. These represent “modular PKSs” in which the functional domains encoded by the DNA sequence are usually ordered parallel to the sequence of reactions carried out on the growing polyketide chain. Type II PKSs are systems made up of individual enzymes, in which each catalytic site is used repeatedly during the biosynthetic process. 
     Genetic studies on prokaryotic PKSs have focused on gram positive microorganisms, particularly on actinomycetes. Myxobacteria are gram negative bacteria that produce a large number of secondary metabolites, including polyketides.  Myxococcus xanthus  produces TA (Rosenberg, et al., 1973; Rosenberg. et al., 1984), which is an antibacterial antibiotic. 
     The polyketide antibiotic Tel-Aviv (hereinafter TA) (Rosenberg, et al., 1973) is synthesized by the gram negative bacterium  Myxococcus xanthus  in a unique multi-step process incorporating a glycine molecule into the polyketide carbon chain, which is elongated through the condensation of 11 acetate molecules by a type I polyketide synthase PKSs). 
     The antibiotic TA was crystallized and its chemical properties were determined. It is a macrocyclic polyketide synthesized through the incorporation of acetate, methionine, and glycine. It inhibits cell wall synthesis by interfering with the polymerization of the lipid-disaccharide-pentapeptide and its ability to adhere avidly to tissues and inorganic surfaces makes it potentially useful in a wide range of clinical applications, such as treating gingivitis. 
     A growing interest in the study of PKS systems and peptide synthetase systems stems from the need to develop new potent biologically active compounds. The use of combinatorial genetics in both systems (PKS and peptide synthetase) separately has led to the production of new polyketides and new peptides. 
     It would therefore by useful to be able to generate new biological agents from secondary metabolites of the antibiotic TA. 
     SUMMARY OF THE INVENTION 
     According to the present invention, there is provided a purified, isolated and cloned DNA sequence partially encoding a functional portion of a polypeptide component required for the synthesis of antibiotic TA. Also provided are purified, isolated and cloned DNA sequences encoding a polypeptide component required for postmodification of antibiotic TA and encoding a gene product involved in the regulation of the biosynthesis of antibiotic TA. A purified, isolated and cloned DNA sequence having a DNA sequence (Seq. ID NO:2 and 20) encoding a polypeptide component required for encoding the TA gene cluster and any mutations thereof is provided. Also provided are methods of using the TA gene cluster and any mutations thereof is provided. Also provided are methods of using the TA genes for combinatorial genetics and of using the TA genes encoding for synthesis and modification or regulation of antibiotic TA. 
    
    
     
       DESCRIPTION OF THE DRAWING 
       Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawing wherein: 
         FIG. 1  shows the physical maps of the DNA regions involved in TA synthesis. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention consists of DNA sequences isolated from  Myxococcus xanthus  TA gene cluster of at least 42 kb, encoding peptides involved in TA production, as best shown in Seq ID NOs: 1 and 3-19. The TA gene cluster has been purified, isolated and cloned. The purification, isolation and cloning was done according to the methods described in Marshak et al, “Strategies for Protein Purification and Characterization. A laboratory course manual” CSHL Press, 1966. 
     A DNA fragment of at least 42 kb (FIG.  1 ), encoding genes involved in TA production in  Myxococus xanthus  has been identified, cloned and analyzed. These steps were done in accordance with Marshak et al, “Strategies for Protein Purification and Characterization. A laboratory course manual” CSHL Press, 1966. This fragment consists a large region (designated Region 2) of about 20 kb, encoding the polypeptides TaA, TaB, TaC, TaD, TaE, TaF, TaG, TaH, TaI, TaJ, TaK, TaL, TaM, TaN, TaR3, TaR2 and TaR1, which are responsible for the regulation of the post-modification of TA. An additional fragment (designated Region 1) of approximately 8-10 kb is located 10-20 kb downstream of the post modification region, encoding the Ta1 polypeptide. The Ta1 polypeptide is involved in the incorporation of the glycine into the TA polypeptides chain. This novel polypeptide is made up of a peptide synthetase unit lying between two PKS modules. 
     The potential of this unique polypeptide in combining the two systems can lead to a new family of compounds, emerging from various combinations which can be utilized for combinatorial genetics. Such utilization can produce, for example, new bioactive agents, new polyketides and new peptides. Additionally, the TA gene cluster can be utilized in a method for the synthesis, modification or regulation of the TA antibiotic. 
     
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Polypeptides encoded by the TA gene cluster of  Myxococcus xanthus   
               
             
          
           
               
                 SEQ ID NO. 
                 Function 
               
               
                   
               
             
          
           
               
                 1 
                 Ta1—synthetase unit and a PKS module 
               
               
                 3 
                 TaR1—a surface layer protein 
               
               
                 4 
                 TaR2—two component system, response regulator 
               
               
                 5 
                 TaR3—two component system, kinase sensor 
               
               
                 6 
                 TaA—NUS-G like transcription antiterminator 
               
               
                 7 
                 TaB—an ACP 
               
               
                 8 
                 TaC—beta-ketoacyl (ACP) synthase III (KAS III FabH) 
               
               
                 9 
                 TaD—membrane associated protein 
               
               
                 10 
                 TaE—an ACP 
               
               
                 11 
                 TaF—beta-ketoacyl (ACP) synthase III (KAS III FabH) 
               
               
                 12 
                 TaG—signal peptidase II (LSPA) 
               
               
                 13 
                 TaH—cytochrome P450 hydroxylase (cP450) 
               
               
                 14 
                 TaI—malonyl CoA (ACPP transacylase (MCT, FabD) 
               
               
                 15 
                 TaJ—malonyl CoA (ACPP transacylase (MCT, FabD) 
               
               
                 16 
                 TaK—3-oxoacyl (ACP) synthase (KAS I, FabB) 
               
               
                 17 
                 TaL—enoyl CoA hydratase 
               
               
                 18 
                 TaM—enoyl CoA hydratase 
               
               
                 19 
                 TaN—O-methyltransferase (fragment) 
               
               
                   
               
               
                 PKS—polyketide synthase  
               
               
                 ACP—acyl carrier protein  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 DNA sequences identified from the TA gene cluster of 
               
               
                 
                   Myxococcus xanthus 
                 
               
             
          
           
               
                   
                 SEQ ID NO. 
                 Description 
               
               
                   
                   
               
             
          
           
               
                   
                 2 
                 Nucleotides 1-7178  
               
               
                   
                 20 
                 Nucleotides 1-19053 
               
               
                   
                   
               
             
          
         
       
     
     Mutations imparting defects into the TA gene cluster can be point mutations, deletions or insertions. The mutations can occur within the nucleotide sequence of the allele of the TA gene cluster such that the resulting amino acid sequence of the TA gene cluster product is altered. 
     In one embodiment of the present invention, the TA gene cluster can be included in a vector or recombinant expression vector. This vector containing the TA gene cluster is able to transform a suitable eucaryotic or procaryotic host cell. A suitable host cell can be determined by one skilled in the art. An example of a suitable cell which can be transformed by the TA gene cluster is an  E. coli  cell. 
     In another embodiment of the present invention, the a DNA fragment encoding the TA gene cluster can be cloned into a cosmid, as shown in FIG.  1 . This DNA fragment contains a large region of about 20 kb, encoding the genes responsible for the regulation and the post-modification of TA. An additional fragment of approximately eight to ten kb is located 10-20 kb downstream of the post-modification region and encodes the enzyme responsible for the incorporation of the glycine into the polyketide chain. The novel polyketide chain is made up of a peptide synthetase unit lying between two PKS modules (See FIG.  1 ). 
     The above discussion provides a factual basis for the use of the TA gene cluster. The methods used with and the utility of the present invention can be shown by the following non-limiting examples and accompanying figure. 
     EXAMPLES 
     General Methods 
     Methods: 
     General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al.,  Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley and Sons, Baltimore, Md. (1989). Polymerase chain reaction (PCR) is carried out generally as in  PCR Protocols: A Guide To Methods And Applications , Academic Press, San Diego, Calif. (1990). Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, are performed as generally described in Sambrook et al., 1989 , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. In-situ (In-cel) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al, 1996, Blood 87:3822.) 
     Recombinant Protein Purification 
     Marshak et al, “Strategies for Protein Purification and Characterization. A laboratory course manual.” CSHL Press, 1996. 
     Example 1 
     Analysis of the TA Gene Cluster by Chromosomal Restriction Map. 
     Chromosomal DNA of several transposition mutants (ER-2514, ER-1037, ER-1030, ER-1311, ER-7513, ER-3708, ER-4639 and ER-6199; Varon et al., 1992) was extracted, digested with restriction enzymes that cut within the transposon, and analyzed by Southern hybridization with six different probes (originating from TnV and Tn5lac). We used probes designed to hybridize either to the entire transposon, or to its 5′ or 3′ ends. A chromosomal restriction map of the whole gene cluster was constructed on the basis of these results (FIG.  1 ). The data refined the transduction map (Varon et al, 1992) and further indicated that all the genes in the cluster are transcribed in the same direction (see FIG.  1 ). 
     Preparation of TA-specific Probes 
     DNA from the TnV mutant ER-4639, ER1311 and ER-6199 was digested with KpnI (does not restrict TnV), self-ligated and transformed into  E. coli  XL1-Blue MR using the transposon-derived kanamycin resistance for selection. Tranformant clones pPYT4639, pPYT1311/p5 and pPYT6199 carried a 1.5 kb, 2.3 kb and a 11.2 kb fragment, respectively (see FIG.  1 ). 
     Cloning of a  M. Xanthus  DNA Region Encoding Genes Involved in TA Biosynthesis. 
     A library of  M. xanthus  ER-15 was constructed in the cosmid vector SUPERCOS-1 and screened using specific TA probes obtained from transposition mutants (ER-4639, ER-1311 and ER-6199, see map) that contain a TnV transposon. Seventy four recombinant cosmids that carried genes required for TA production were identified through colony hybridization. The cosmids, pPYCC64 and pPYCC44, which hybridized to these probes were further characterized through restriction analysis (see  FIG. 1 ) and sub cloned for sequencing. 
     Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 
     The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. 
     Obviously, many modifications and variations of the present invention are possible in tight of the above teachings. It is, therefore, to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described. 
     References 
     1. Rosenberg, E., Vaks, B. and Zuckerberg. A. Bactericidal action of an antibiotic produced by  Myxococcus xanthus . Antimicrob. Agents. Chemother. 4:507-513 (1973). 
     2. Rosenberg, E., Porter, J. M., Nathan, P. N., Manor, A. and Varon, M. Antibiotic TA: an adherent antibiotic. Bio/Technology. 2:796-799 (1984). 
     3. Varon et al., Mutation and mapping of genes involved in production of the antibiotic TA in  micrococcus xanthus . Antimicrob. Agents Chemother. 36:2316-2321 (1992). 
     4. Marshak et al, “Strategies for Protein Purification and Characterization. A laboratory course manual.” CSHL Press, 1996. 
     5. Testoni et al, 1996, Blood 87:3822. 
     6 . PCR Protocols: A Guide To Methods And Applications , Academic Press, San Diego, Calif. (1990). 
     7. Sambrook et al.,  Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992). 
     8. Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley and Sons, Baltimore, Md. (1989).