Gene conferring resistance to the antibacterial 4,5-dihydroxy-2-cyclopenten-1-one (DHCP), the protein encoded by same, and applications thereof

Abstract

The invention relates to a gene, dep, which confers resistance to the antibacterial activity of 4,5-dihydroxy-2-cyclopenten-1-one (DHCP). The invention further relates to the putative protein encoded by dep, which is a hydrophobic, transmembrane efflux protein specific for DHCP. The invention further relates to plasmids containing the dep gene, and to bacterial cells expressing dep. Furthermore, the invention provides applications for use in conferring resistance to antibacterial activity in organisms. The dep gene can be used to identify compounds which inhibit the efflux activity responsible for the resistance to DHCP or to compounds which are functionally equivalent to DHCP.

We claim:  
     
         1 . A DHCP efflux protein, which is specific for 4,5-dihydroxy-2-cyclopenten-1-one (DHCP).  
     
     
         2 . The protein of  claim 1 , which is a transmembrane protein that forms a cytoplasmic channel specific for efflux transport of DHCP.  
     
     
         3 . The protein of  claim 1 , which confers resistance to DHCP.  
     
     
         4 . The protein of  claim 1 , which protein is from  E. coli.    
     
     
         5 . The protein of  claim 1 , which protein does not confer cross-resistance to the any of the following antibiotics: chloramphenicol, spectinomycin and tetracycline.  
     
     
         6 . The protein of  claim 1 , which protein possesses 13 predicted transmembrane-spanning α-helices.  
     
     
         7 . A gene encoding dep, the DHCP efflux protein.  
     
     
         8 . The dep gene of  claim 7 , wherein the dep gene is from  E. coli.    
     
     
         9 . The gene of  claim 7 , wherein said gene confers resistance to DHCP or a functionally equivalent compound when present in multiple copies in a bacterial cell.  
     
     
         10 . A plasmid comprising the dep gene, which plasmid confers expression of multiple copies of the dep gene in bacteria cells that have been transformed with said plasmid.  
     
     
         11 . The plasmid of  claim 10 , which plasmid confers resistance to DHCP and does not confer cross-resistance to any of the following antibiotics: chloramphenicol, spectinomycin and tetracycline.  
     
     
         12 . Bacteria cells containing multiple copies of the plasmid of  claim 10 .  
     
     
         13 . The bacteria cells of  claim 12 , which bacteria cells are resistant to DHCP.  
     
     
         14 . A method which uses the gene of  claim 7  to identify a compound which inhibits efflux activity responsible for resistance to DHCP or a functionally equivalent compound.