Identification of inhibitors of a bacterial stress response

Abstract

A system, a composition, a method and a kit for identifying anti-bacterial agents are provided. The invention described herein is useful in identifying inhibitors of any bacterial stress response. Moreover, the invention can be applied to any sRNA and its target, any transcription factor and its target, and any transcription factor/sRNA pair (i.e., a transcription factor that regulates a sRNA). In particular, the present invention provides a system, a composition, a method and a kit for the identification of cyclic peptides that block the σ E pathway in Escherichia coli.

1 .- 28 . (canceled) 
     
     
         29 . A bacterium comprising:
 i) a first nucleic acid encoding a transcription factor,   ii) a second nucleic acid encoding a small RNA (sRNA), wherein the second nucleic acid is operably linked to the first nucleic acid, and   iii) a third nucleic acid encoding a target protein, wherein the third nucleic acid encodes a messenger RNA (mRNA) that is capable of being bound by the sRNA.   
     
     
         30 . The bacterium of  claim 29 , wherein the transcription factor is selected from the group consisting of σ E , ompR, and Fnr. 
     
     
         31 . The bacterium of  claim 29 , wherein the sRNA is selected from the group consisting of MicA, MicF, RybB, and FnrS. 
     
     
         32 . The bacterium of  claim 29 , wherein the target protein is selected from the group consisting of ompA, ompF, ompC, ompW, ompX, and SodB. 
     
     
         33 . The bacterium of  claim 29 , wherein the bacterium comprises a Gram-negative bacterium selected from the group consisting of  Escherichia coli, Salmonella enterica serovar typhi, Vibrio cholerae, Burkholderia pseudomallei , and  Yersinia enterocolitica.    
     
     
         34 . The bacterium of  claim 29 , wherein the transcription factor comprises σ E , the sRNA comprises MicA, and the target protein comprises ompA. 
     
     
         35 . The bacterium of  claim 29 , wherein the transcription factor comprises σ E , the sRNA comprises RybB, and the target protein comprises ompC. 
     
     
         36 . The bacterium of  claim 29 , wherein the transcription factor comprises ompR, the sRNA comprises MicF, and the target protein comprises ompF. 
     
     
         37 . The bacterium of  claim 29 , wherein the transcription factor comprises Fnr, the sRNA comprises FnrS, and the target protein comprises SodB. 
     
     
         38 . The bacterium of  claim 29 , wherein the target protein further comprises a reporter protein. 
     
     
         39 . The bacterium of  claim 29 , wherein at least one transcriptional terminator sequence is disposed between the first nucleic acid and second nucleic acid. 
     
     
         40 . The bacterium of  claim 35 , wherein the bacterium comprises  Escherichia coli.    
     
     
         41 . The bacterium of  claim 29 , wherein the first nucleic acid further comprises a first inducible promoter. 
     
     
         42 . The bacterium of  claim 29 , wherein the third nucleic acid further comprises a second inducible promoter. 
     
     
         43 . The bacterium of  claim 38 , wherein the reporter protein comprises a luminescent or fluorescent protein. 
     
     
         44 . The bacterium of  claim 38 , wherein the reporter protein comprises yellow fluorescent protein.