Claims
- 1. An isolated bacterial strain having an increased expression level of a transporter that transports an autoinducer into the strain relative to a wildtype strain, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the Vibrio harveyi LuxQ protein, thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes.
- 2. The strain of claim 1, wherein the strain has been genetically engineered to increase expression of the transporter.
- 3. The strain of claim 1, wherein the transporter comprises at least one polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 37-40 and a sequence having at least 25% amino acid identity as determined through use of FASTA version 3.0t78 with the default parameters to one of SEQ ID NOs.: 37-40.
- 4. The strain of claim 1, wherein the transporter comprises a complex comprising each of the sequences of SEQ ID NOs. 37-40 or sequences having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to each of the sequences of SEQ ID NOs.: 37-40.
- 5. The strain of claim 2, wherein the strain comprises at least one vector from which one or more polypeptides included in the transporter are expressed.
- 6. The strain of claim 1, wherein the strain comprises a mutation that increases expression of the transporter.
- 7. The strain of claim 6, wherein the mutation is in a gene encoding a repressor that reduces expression of the transporter.
- 8. The strain of claim 7, wherein the mutation is in a gene comprising the sequence of SEQ ID NO: 28.
- 9. The strain of claim 8, wherein the mutation is in a gene comprising a sequence having at least 30% identity to SEQ ID NO: 28 as determined through use of BLASTN version 2.0 with the default parameters.
- 10. The strain of claim 7, wherein the mutation is in a gene encoding a polypeptide comprising the sequence of SEQ ID NO: 36.
- 11. The strain of claim 7, wherein the mutation is in a nucleic acid comprising a sequence that hybridizes to a probe comprising at least 30 consecutive nucleotides of SEQ ID NO: 28 in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.
- 12. The strain of claim 7, wherein the mutation is in a polypeptide comprising a sequence having at least 25% identity to SEQ ID NO: 36 as determined through use of FASTA version 3.0t78 with the default parameters.
- 13. The strain of claim 7 further comprising a mutation in a gene that inhibits the production of the autoinducer.
- 14. The strain of claim 13, wherein the mutation is in a luxS gene.
- 15. The strain of claim 14, wherein the mutation is in a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9.
- 16. The strain of claim 13, wherein the mutation is in a nucleic acid comprising a sequence having at least 30% identity as determined through use of BLASTN version 2.0 with the default parameters to a sequence selected from the group consisting of SEQ ID NOs: 1-9.
- 17. The strain of claim 13, further comprising a mutation that inhibits the detection of a second autoinducer that is an acylho-moserine lactone.
- 18. The method of claim 17, wherein the second autoinducer is an acyl-homoserine lactone.
- 19. The method of claim 17, wherein the second autoinducer is N-(3-hydroxyacyl)-L-homoserine lactone and the acyl group comprises 4-12 carbon atoms.
- 20. The method of claim 19, wherein the acyl group comprises four carbon atoms.
- 21. The strain of claim 17, wherein the mutation is in the luxN gene.
- 22. The strain of claim 1, wherein the autoinducer is the autoinducer-2 produced by Vibrio harveyi.
- 23. The strain of claim 1, wherein the autoinducer is a pentanedione.
- 24. The strain of claim 20, wherein the pentanedione is 4,5-dihydroxy-2,3-pentanedione.
- 25. The strain of claim 1, wherein the strain belongs to a species selected from the group consisting of S. typhimurium and E. coli.
- 26. The strain of claim 1, wherein the strain belongs to a species selected from the group consisting of Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi and Vibrio cholerae.
- 27. The strain of claim 1, wherein the strain is a strain of Vibrio harveyi.
- 28. A method for identifying a compound that modulates the response to a first autoinducer that is not an acyl-homoserine lactone and that can interact with the Vibrio harveyi LuxQ protein, thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes, comprising:
obtaining a cell having increased expression of a transporter that transports the autoinducer into the cell, wherein the cell produces a detectable signal in response to the first autoinducer; measuring the response of the cell to the first autoinducer in the presence and absence of a test compound; and comparing the responses to determine whether the test compound modulates the response to the first autoinducer.
- 29. The method of claim 28, wherein the cell has been genetically engineered to increase expression of the transporter.
- 30. The method of claim 28, wherein the transporter comprises at least one polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 37-40 and a sequence having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to one of SEQ ID NOs.: 37-40.
- 31. The method of claim 28, wherein the transporter comprises a complex comprising each of the amino acid sequences of SEQ ID NOs. 37-40 or amino acid sequences having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to each of the sequences of SEQ ID NOs.: 37-40.
- 32. The method of claim 29, wherein the cell comprises at least one vector from which one or more polypeptides included in the transporter are expressed.
- 33. The method of claim 29, wherein the cell comprises a mutation that increases expression of the transporter.
- 34. The method of claim 33, wherein the mutation is in a gene encoding a repressor that reduces expression of the transporter.
- 35. The method of claim 34 wherein the mutation is in a gene comprising the sequence of SEQ ID NO: 28.
- 36. The method of claim 34, wherein the mutation is in a gene comprising a sequence having at least 30% identity to SEQ ID NO: 28 as determined through use of BLASTN version 2.0 with the default parameters.
- 37. The method of claim 34, wherein the mutation is in a gene encoding a polypeptide comprising the sequence of SEQ ID NO: 36
- 38. The method of claim 34, wherein the mutation is in a nucleic acid comprising a sequence that hybridizes to a probe comprising at least 20 consecutive nucleotides of SEQ ID NO: 28 in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.
- 39. The method of claim 34, wherein the mutation is in a polypeptide comprising a sequence having at least 25% identity to SEQ ID NO: 36 as determined through use of FASTA version 3.0t78 with the default parameters.
- 40. The method of claim 34 wherein the cell further comprises a mutation in a gene that inhibits the production of the autoinducer.
- 41. The method of claim 40, wherein the mutation that inhibits the production of the autoinducer is in a luxS gene.
- 42. The method of claim 41, wherein the mutation that inhibits the production of the autoinducer is in a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-9.
- 43. The method of claim 40, wherein the mutation is in a nucleic acid comprising a sequence having at least 30% nucleotide identity as determined through use of BLASTN version 2.0 with the default parameters to a sequence selected from the group consisting of SEQ ID NOs: 1-9.
- 44. The method of claim 40, wherein the cell further comprises a mutation that inhibits the detection of a second autoinducer that is an acylhomoserine lactone.
- 45. The method of claim 44, wherein the second autoinducer is N-(3-hydroxyacyl)-L-homoserine lactone and the acyl group comprises 4-12 carbon atoms.
- 46. The method of claim 45, wherein the acyl group comprises four carbon atoms.
- 47. The method of claim 44, wherein the mutation that inhibits the detection of the second autoinducer is in the luxN gene.
- 48. The method of claim 28, wherein the autoinducer is the autoinducer-2 produced by Vibrio harveyi.
- 49. The method of claim 28, wherein the autoinducer is a pentanedione.
- 50. The method of claim 49, wherein the pentanedione is 4,5-dihydroxy-2,3-pentanedione.
- 51. The method of claim 28, wherein the cell belongs to a species selected from the group consisting of S. typhimurium and E. coli.
- 52. The method of claim 28, wherein the cell belongs to a species selected from the group consisting of Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi and Vibrio cholerae.
- 53. The method of claim 1, wherein the cell is a Vibrio harveyi cell.
- 54. A method for screening a candidate compound for the ability to bind to a transporter that transports an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the Vibrio harveyi LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes, comprising:
contacting the transporter with the candidate compound; and determining whether the compound specifically binds to the transporter.
- 55. The method of claim 54, wherein the compound comprises a detectable label.
- 56. The method of claim 54, wherein the transporter comprises at least one polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.: 37-40 and a sequence having at least 25% amino acid identity as determined through use of FASTA version 3.0t78 with the default parameters to one of SEQ ID NOs.: 37-40.
- 57. The method of claim 54, wherein the transporter comprises a complex comprising each of the amino acid sequences of SEQ ID NOs. 37-40 or amino acid sequences having at least 25% amino acid identity as determined through use of FASTA version 3.0t78 with the default parameters to each of the amino acid sequences of SEQ ID NOs.: 37-40.
- 58. A method of screening a candidate compound for the ability to modulate the binding of an autoinducer to a transporter, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the Vibrio harveyi LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes, comprising:
comparing the binding of the autoinducer to the transporter in the presence and absence of the candidate compound; and determining whether the the extent of binding of the autoinducer to the transporter in the presence of the compound increases or decreases relative to the extent of binding in the absence of the compound.
- 59. A method of screening a candidate compound for the ability to bind a polypeptide comprising:
contacting a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 37-40 and a sequence having at least 25% identity as determined through use of FASTA version 3.0t78 with the default parameters to one of the sequences of SEQ ID NOs.: 37-40 with the compound; and determining whether the compound specifically binds to the polypeptide.
- 60. An isolated bacterial strain comprising a mutation that inhibits the transport of an autoinducer that is not an acyl-homoserine lactone and that can interact with the Vibrio harveyi LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes.
- 61. The bacterial strain of claim 60, wherein the mutation is in a gene selected from the group consisting of the lsrA, lsrB, lsrC, and lsrD genes.
- 62. The bacterial strain of claim 61, wherein the strain is a Salmonella typhimurium strain.
- 63. The bacterial strain of claim 62, wherein the mutation is in a sequence selected from the group consisting of SEQ ID NOs.: 29-32.
- 64. The bacterial strain of claim 63, wherein the mutation is in a gene encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs.: 37-40.
- 65. The bacterial strain of claim 63, wherein the mutation is in a nucleic acid comprising a sequence selected from the group consisting of a sequence having at least 30% nucleotide identity to one of SEQ ID NOs.: 29-32 as determined through use of BLASTN version 2.0 with the default parameters.
- 66. A bacterial strain that overexpresses or underexpresses a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 36-43 and a sequence having at least 25% identity to one of SEQ ID NOs: 36-43 relative to a wildtype strain.
- 67. A bacterial strain that overexpresses or underexpresses a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs. 36-43 and a sequence having at least 25% identity to one of SEQ ID NOs: 36-43 relative to a wildtype strain.
- 68. An isolated or purified nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs.: 28-35 and a fragment comprising at least 20 consecutive nucleotides of one of of SEQ ID NOs.: 28-35.
- 69. An isolated or purified nucleic acid comprising a fragment of one of SEQ ID NOs.: 28-35 that encodes a polypeptide that can facilitate the transport of an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the Vibrio harveyi LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes.
- 70. A recombinant vector comprising a sequence selected from the group consisting of SEQ ID NOs: 28-35 operably linked to a heterologous promoter.
- 71. An isolated or purified protein comprising a sequence selected from the group consisting of SEQ ID NOs.: 36-43 and a fragment comprising at least 10 consecutive amino acids of one of of SEQ ID NOs. 36-43.
- 72. An isolated or purified polypeptide comprising a fragment of one of SEQ ID NOs.: 36-43 that encodes a polypeptide that can facilitate the transport of an autoinducer into a cell, wherein the autoinducer is not an acyl-homoserine lactone and can interact with the Vibrio harveyi LuxQ protein thereby inducing expression of a Vibrio harveyi operon comprising the luxCDABE genes.
- 73. An antibody that binds to a polypeptide selected from the group consisting of SEQ ID NOs.: 36-43.
RELATED APPLICATION INFORMATION
[0001] This application claims priority to U.S. Provisional Application No. 60/336,324, filed on Oct. 26, 2001 and entitled Inhibitors of Autoinducer Transporters, the disclosure of which is hereby incorporated by reference in its entirety.
STATEMENT OF GOVERNMENT INTEREST
[0002] The National Science Foundation Grants MCB-0083160, MCB-0094447, and. MCB-9506033 and The Office of Naval Research Grant Number N00014-99-0767 supported this work. Accordingly, the U.S. Government has certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60336324 |
Oct 2001 |
US |