Method of selecting recombinant DNA-containing streptomyces

Abstract

A novel method of selecting Streptomyces recombinant DNA-containing host cells and vectors useful in the method are described. The vectors confer tylosin resistance to sensitive Streptomyces host cells and thus provide a convenient method of selecting Streptomyces transformants. The novel tylosin resistance-conferring gene described can be isolated on an ˜2.6 kb KpnI restriction fragment from plasmid pSVB2. Plasmid pSVB2 can be isolated from Streptomyces lividans TK23/pSVB2 (NRRL 15880).

We claim: 
     
       1. A method for selecting a recombinant DNA-containing Streptomyces host cell, said method comprising: (a) transforming a tylosin-sensitive, restrictionless Streptomyces host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Streptomyces host cell, said vector comprising a DNA sequence that confers resistance to tylosin, and   (b) culturing said transformed cell under growth conditions suitable for selection for tylosin resistance, subject to the limitation that said host cell is susceptible to transformation, cell division and culture.     
     
     
       2. The method of claim 1 wherein the recombinant DNA cloning vector is a plasmid. 
     
     
       3. The method of claim 1 wherein the recombinant DNA cloning vector is a phage. 
     
     
       4. The method of claim 2 wherein the plasmid is selected from the group consisting of pSVB2, pSVB12, pSVB23, pSVB16, pSVB18, pSVB20 and pSVB22. 
     
     
       5. The method of claim 2 wherein the plasmid is pSVB2. 
     
     
       6. The method of claim 2 wherein the plasmid is pSVB12. 
     
     
       7. The method of claim 2 wherein the plasmid is pSVB23. 
     
     
       8. The method of claim 2 wherein the plasmid is pSVB16. 
     
     
       9. Themethod of claim 2 wherein the plasmid is pSVB18. 
     
     
       10. The method of claim 2 wherein the plasmid is pSVB20. 
     
     
       11. The method of claim 2 wherein the plasmid is pSVB22. 
     
     
       12. The method of claim 3 wherein the phage is selected from the group consisting of pSVB3310 and pSVB3311. 
     
     
       13. The method of claim 1 wherein the transformed Streptomyces host cell is selected from the group consisting of Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces griseofuscus, Streptomyces lividans, Streptomyces cinnamonensis and Streptomyces .[.toyocaenisis.]. .Iadd.toyocaensis. .Iaddend. 
     
     
       14. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB2. 
     
     
       15. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB12. 
     
     
       16. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB23. 
     
     
       17. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB16. 
     
     
       18. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB18. 
     
     
       19. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB20. 
     
     
       20. The method of claim 13 wherein the transformed host cell is Streptomyces griseofuscus/pSVB22. 
     
     
       21. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB2. 
     
     
       22. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB12. 
     
     
       23. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB23. 
     
     
       24. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB16. 
     
     
       25. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB18. 
     
     
       26. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB20. 
     
     
       27. The method of claim 13 wherein the transformed host cell is Streptomyces lividans/pSVB22. 
     
     
       28. A recombinant DNA cloning vector capable of autonomous replication or integration in a Streptomyces host cell, said vector comprising a DNA sequence that confers resistance to tylosin in a tylosin-sensitive Streptomyces host cell. 
     
     
       29. A vector .Iadd.of .Iaddend.claim 28 which is selected from the group consisting of plasmids pSVB2, pSVB12, pSVB23, pSVB16, pSVB18, pSVB20 and pSVB22; and phages pSVB3310 and pSVB3311. 
     
     
       30. The vector of claim 29 that is plasmid pSVB2. 
     
     
       31. The vector of claim 29 that is plasmid pSVB12. 
     
     
       32. The vector of claim 29 that is plasmid pSVB23. 
     
     
       33. The vector of claim 29 that is plasmid pSVB16. 
     
     
       34. The vector of claim 29 that is plasmid pSVB18. 
     
     
       35. The vector of claim 29 that is plasmid pSVB20. 
     
     
       36. The vector of claim 29 that is plasmid pSVB22. 
     
     
       37. The vector of claim 29 that is plasmid pSVB3310. 
     
     
       38. A Streptomyces host cell transformed by a vector of claim 28, said Streptomyces host cell being tylosin-sensitive when not transformed with said vector. 
     
     
       39. The Streptomyces host cell of claim 38 transformed by a vector from the group consisting of plasmids pSVB2, .[.L pSVB12,.]. .Iadd.pSVB12, .Iaddend.pSVB23, pSVB16, pSVB18, pSVB20 and pSVB22, and phage pSVB3310 and pSVB3311. 
     
     
       40. The transformed Streptomyces host cell of claim 39 that is Streptomyces ambofaciens. 
     
     
       41. The transformed Streptomyces host cell of claim 39 that is Streptomyces lividans. 
     
     
       42. The transformant of claim 40 that is Streptomyces ambofaciens/pSVB2. 
     
     
       43. The transformant of claim 41 that is Streptomyces lividans/pSVB3310. 
     
     
       44. A method for selecting a recombinant DNA-containing Nocardia host cell, said method comprising: (a) transforming a tylosin-sensitive, restrictionless Nocardia host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Noardia host cell, said vector comprising a DNA sequence that confers resistance to tylosin, and   (b) culturing said transformed cell under growth conditions suitable for selection for tylosin resistance, subject to the limitation that said host cell is susceptible to transformation, cell division and culture.     
     
     
       45. The method of claim 44 wherein the recombinant DNA cloning vector is a plasmid. 
     
     
       46. A Nocardia host cell transformed by a recombinant DNA cloning vector capable of autonomous replication or integration in said Nocardia host cell, said vector comprising a DNA sequence that confers resistance to tylosin in a tylosin-sensitive Nocardia host cell, said Nocardia host cell being tylosin-sensitive when not transformed with said vector. .Iadd. 
     
     
       47.  A recombinant DNA comprising a DNA sequence capable of conferring tylosin resistance on a Streptomyces or Nocardia host cell. .Iaddend. .Iadd.48. The recombinant DNA of claim 47 wherein said DNA sequence is 
     
     
        isolated from Streptomyces fradiae. .Iaddend. .Iadd.49.  A recombinant DNA cloning vector comprising the DNA fragment of claim 48. .Iaddend. .Iadd.50. A Streptomyces host cell transformed by a vector of claim 49. .Iaddend. .Iadd.51. A DNA fragment of claim 48 which is a tylosin resistance-conferring restriction fragment of plasmid pSVB2. .Iaddend. .Iadd.52. A recombinant DNA cloning vector comprising the DNA fragment of claim 51. .Iaddend. .Iadd.53. A Streptomyces host cell transformed by a vector of claim 52. .Iaddend. .Iadd.54. The DNA fragment of claim 51 which is the ˜2.6 kb KpnI restriction fragment of plasmid pSVB2. .Iaddend. .Iadd.55. A recombinant DNA cloning vector comprising the DNA fragment of claim 54. .Iaddend. .Iadd.56. A Streptomyces host cell transformed by a vector of claim 55. .Iaddend.