Method for identifying microbial proliferation genes

• US 6,228,579 B1
• Filed: 11/14/1997
• Issued: 05/08/2001
• Est. Priority Date: 11/14/1997
• Status: Expired due to Fees

• Current Assignee: San Diego State University Foundation
• Original Assignee: San Diego State University Foundation
• Inventors: Zyskind, Judith W., Forsyth, R. Allyn
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/971,090
• Time in Patent Office: 1,271 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/24.33
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218

Abstract

A method for identifying endogenous microbial proliferation genes for growth and viability is disclosed herein. The method involves exogenous nucleic acids that are used to conditionally produce antisense inhibitors of endogenous complementary mRNAs in a microorganism. Antisense fragments that result in lethality when expressed indicate that the endogenous gene is a proliferation gene. The method can also be used with sequences in sense orientation. The strategy can be used to identify new gene targets for novel antibiotics.

Claims

1. A method for identifying microbial proliferation genes, comprising the steps of:

a) introducing an exogenous nucleic acid into a microorganism, the exogenous nucleic acid having substantial sequence identity to an endogenous microbial gene and wherein the exogenous nucleic acid is a random fragment or a random sequence; and

b) identifying the endogenous gene as a microbial proliferation gene by comparing the proliferation or viability of the microorganism when the exogenous nucleic acid is expressed in or introduced into the microorganism with the proliferation or viability of the microorganism when the exogenous nucleic acid is not present or not expressed.

2. The method of claim 1, wherein the exogenous nucleic acid is operably linked in antisense orientation to the regulatory element.

3. The method of claim 1, wherein the exogenous nucleic acid is obtained from total chromosomal and episomal genomic DNA or RNA or cDNA or synthetic DNA.

4. The method of claim 1, wherein the exogenous nucleic acid is present on a vector.

5. The method of claim 1, wherein the microorganism is a bacterium.

6. The method of claim 5, wherein the bacterium is a pathogen.

7. The method of claim 6, wherein the bacterium is a human pathogen.

8. The method of claim 6, wherein the bacterium is an animal pathogen.

9. The method of claim 5, wherein the bacterium is a gram-negative bacterium.

10. The method of claim 9, wherein the bacterium is Escherichia coli.

11. The method of claim 5, wherein the bacterium is a gram-positive bacterium.

12. The method of claim 11, wherein the bacterium is Staphylococcus aureus.

13. The method of claim 1, wherein the microorganism is a fungus.

14. The method of claim 13, wherein the microorganism is a yeast.

15. The method of claim 1, wherein the microorganism is an Archaebacteria.

16. The method of claim 1, wherein the exogenous nucleic acid is from about 10 to about 5,000 nucleotides in length.

17. The method of claim 16, wherein the exogenous nucleic acid is from about 15 to about 1,500 nucleotides in length.

18. The method of claim 6, wherein the bacterium is a plant pathogen.

23. The method of claim 1 or claim 19, wherein the exogenous nucleic acid is operably linked to a regulatory element effective for controlling expression of the exogenous nucleic acid.

24. The method of claim 23, wherein the regulatory element effective for controlling expression of the exogenous nucleic acid is an inducible regulatory sequence.

25. The method of claim 24, wherein the inducible regulatory sequence that controls expression of the exogenous nucleic acid is a promoter induced in response to a chemical inducer.

26. The method of claim 24, wherein the inducible regulatory sequence that controls expression of the exogenous nucleic acid is a promoter induced in response to a environmental changes.

27. The method of claim 23, wherein the regulatory element effective for controlling expression of the exogernous nucleic acid is a promoter repressed in response to the presence of a chemical agent.

28. The method of claim 1 or 19, wherein the random fragment is generated by physical shearing or by partial or total restriction digestion.

29. The method of claim 1 or 19, wherein the random sequence is synthetic DNA.

30. The method of claim 1 or 19, wherein comparing the proliferation of the microorganism when the exogenous gene is expressed in or introduced into the microorganism with the proliferation of the microorganism when the exogenous nucleic acid is not present or not expressed is determined by comparative or replica plating of the microorganism.

31. The method of claim 1 or 19, wherein the effect on microbial proliferation is determined by comparing the effect of expression of the exogenous nucleic acid to respiratory activity as compared to the respiratory activity of the microorganism when the exogenous nucleic acid is not present or not expressed.

32. The method of claim 1 or 19, wherein comparing the proliferation of the microorganism when the exogenous gene is expressed in or introduced into the microorganism with the proliferation of the microorganism when the exogenous nucleic acid is not present or not expressed is determined by measuring colony-forming units, measuring light scattering or optical density, measuring the number of organisms in a particle counter, measuring the fluorescence of cell cultures or of individual cells after addition of fluorescent dyes, measuring the incorporation of precursors to macromolecules, or measuring the uptake of metabolites.

33. The method of claim 1 or 19, wherein the exogenous nucleic acid is operably linked in a sense orientation.

34. The method of claim 33, wherein the exogenous nucleatic acid expresses sense RNA.

35. The method of claim 34, wherein the sense RNA encodes a protein or a truncated peptide that interferes with the normal function of the endogenous proliferation product.

36. The method of claim 35, wherein the protein or peptide has a dominant lethal effect.

19. A method for identifying a proliferation gene in a pathogenic microorganism, comprising the steps of:

a) introducing an exogenous nucleic acid into the pathogenic microorganism, the exogenous nucleic acid having substantial sequence identity to an endogenous gene in the pathogenic microorganism, wherein the exogenous nucleic acid is a random fragment or a random sequence; and

b) identifying the endogenous gene as a proliferation gene by comparing proliferation of the pathogenic microorganism when the exogenous nucleic acid is expressed with proliferation of the pathogenic microorganism when the exogenous nucleic acid is not present or expressed, wherein reduced proliferation of the pathogenic microorganism in the presence of the exogenous nucleic acid is indicative of the proliferation gene.

20. The method of claim 19, wherein the pathogenic microorganism is a pathogen of a mammal.

21. The method of claim 20, wherein the mammal is a human.

22. The method of claim 19, wherein the pathogenic microorganism is a pathogen of a plant.