Multiple antibiotic resistance operon assays

Abstract

An isolated and cloned region of a bacterial chromosome containing a multiple antibiotic resistance operon is disclosed. A description of the structure and function of the operon is provided as are assorted recombinant DNA constructs involving the operon or fragments thereof. The diagnostic, therapeutic and experimental uses of these constructs are also disclosed. Methods of evaluating the antibiotic effectiveness of compositions are disclosed and methods of treatment employing effective compositions are provided.

1 . A method for predicting the antibiotic effectiveness of a composition comprising exposing bacteria to said composition and assaying the effect of said exposure on the expression of a genetic locus wherein said expression is regulated at least in part and at least indirectly by at least a functional fragment of a regulatory region of a bacterial multiple antibiotic resistance operon within said bacteria.  
     
     
         2 - 85 . (canceled)  
     
     
         86 . A method for screening the ability of a composition to inhibit growth of a microbe in a host comprising contacting the microbe with the composition and determining the effect of the composition on the level of expression or the activity of a protein, wherein the protein is structurally and functionally related to MarA.  
     
     
         87 . A method for screening the ability of a composition to inhibit growth of a microbe in a host comprising contacting the microbe with the composition and determining the effect of the composition on the level of expression or the activity of a protein that promotes multiple antibiotic resistance in the microbe.  
     
     
         88 . The method of  claim 86 , wherein the protein is selected from the group consisting of: araC, rhaR, rhaS, melR, virF, xylS, rns, soxS, and marA.  
     
     
         89 . The method of  claim 86 , wherein the protein is a mar enhancer.  
     
     
         90 . The method of  claim 86 , wherein the protein is marA.  
     
     
         91 . The method of  claim 86 , wherein the protein is soxS.  
     
     
         92 . The method of  claim 86 , wherein the protein is araC.  
     
     
         93 . The method of  claim 86 , wherein the protein is rhaR.  
     
     
         94 . The method of  claim 86 , wherein the protein is rhaS.  
     
     
         95 . The method of  claim 86 , wherein the protein is melR.  
     
     
         96 . The method of  claim 86 , wherein the protein is virF.  
     
     
         97 . The method of  claim 86 , wherein the protein is xylS.  
     
     
         98 . The method of  claim 86 , wherein the protein is rns.  
     
     
         99 . The method of  claim 86 , wherein the activity of the protein is determined.  
     
     
         100 . The method of  claim 86 , wherein the level of expression of the protein is determined.  
     
     
         101 . The method of  claim 99 , wherein the step of determining the activity of the protein is performed by determining the level of transcription of a regulatory region of an operon whose transcription is regulated by the protein.  
     
     
         102 . The method of  claim 101 , wherein the regulatory region of an operon is micF or ompF.  
     
     
         103 . The method of  claim 101 , wherein the step of determining the level of transcription of gene whose transcription is regulated by the protein comprises measuring the level of a product produced from a marker locus operably linked to the regulatory region of an operon whose transcription is regulated by the protein.  
     
     
         104 . The method of  claim 103 , wherein the marker gene is lacZ.  
     
     
         105 . The method of  claim 103 , wherein the marker gene is phoA.  
     
     
         106 . The method of  claim 86 , wherein the protein is present in a bacterium of a genus selected from the group consisting of:  Escherichia, Erwinia, Pseudomonas, Yersinia, Klebsiella, Serratia, Citrobacter, Enterobacter, Hafnia, Salmonella , and  Shigella.