Methods and composition for the identification of antibiotics that are not susceptible to antibiotic resistance

Abstract

Compositions are provided to identify functional mutant ribosomes that may be used as drug targets. The compositions allow isolation and analysis of mutations that would normally be lethal and allow direct selection of rRNA mutants with predetermined levels of ribosome function. The compositions of the present invention may be used to identify antibiotics to treat a large number of human pathogens through the use of genetically engineered rRNA genes from a variety of species. The invention further provides novel plasmid constructs to be used in the methods of the invention.

What is claimed: 
     
         1 . A plasmid comprising an rRNA gene having a mutant Anti-Shine-Dalgarno sequence, at least one mutation in said rRNA gene, and a genetically engineered gene which encodes a selectable marker having a mutant Shine-Dalgarno sequence, wherein the mutant Anti-Shine-Dalgarno and the mutant Shine-Dalgarno sequence are a mutually compatible pair. 
     
     
         2 . The plasmid of  claim 1 , wherein the rRNA gene is from a species selected from the group consisting of  Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella typhi, Yersenia pestis, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Chlamydia trachomatis, Saccharomyces cerevesiae, Candida alhicans , and trypanosome. 
     
     
         3 . The plasmid of  claim 1 , wherein the selectable marker is chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), or both CAT and GFP. 
     
     
         4 . The plasmid of  claim 1 , wherein the mutant Anti-Shine-Dalgarno sequence is selected from the group consisting of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, and 159. 
     
     
         5 . The plasmid of  claim 1 , wherein the mutant Shine-Dalgarno sequence is selected from the group consisting of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158. 
     
     
         6 . The plasmid of  claim 4 , wherein the mutant Anti-Shine-Dalgarno sequence and the mutant SD sequence are a mutually compatible pair. 
     
     
         7 . The plasmid of  claim 6 , wherein the mutually compatible mutant Shine-Dalgarno and mutant Anti-Shine-Dalgarno pair permits translation by the rRNA of the selectable marker. 
     
     
         8 . The plasmid of  claim 3 , wherein the selectable marker is CAT. 
     
     
         9 . The plasmid of  claim 3 , wherein the selectable marker is GFP. 
     
     
         10 . A cell comprising the plasmid of  claim 1 . 
     
     
         11 . The cell of  claim 10 , wherein the mutations in the rRNA gene affect the quantity of selectable marker produced. 
     
     
         12 . The cell of  claim 10 , wherein the cell is a bacterial cell. 
     
     
         13 . The plasmid of  claim 1 , wherein the DNA sequence encoding the rRNA gene is under the control of an inducible promoter. 
     
     
         14 . A plasmid comprising an  E. coli  16S rRNA gene having a mutant Anti-Shine-Dalgarno sequence, at least one mutation in said 16S rRNA gene, and a genetically engineered gene which encodes GFP having a mutant Shine-Dalgarno sequence, wherein the mutant Anti-Shine-Dalgarno and the mutant Shine-Dalgarno sequence are a mutually compatible pair. 
     
     
         15 . The plasmid of  claim 14 , wherein the mutant Anti-Shine-Dalgarno sequence is selected from the group consisting of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, and 159. 
     
     
         16 . The plasmid of  claim 14 , wherein the mutant Shine-Dalgarno sequence is selected from the group consisting of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158. 
     
     
         17 . The plasmid of  claim 15 , wherein the mutant Anti-Shine-Dalgarno sequence and the mutant Shine-Dalgarno sequence are a mutually compatible pair. 
     
     
         18 . The plasmid of  claim 17 , wherein the mutually compatible mutant Shine-Dalgarno and mutant Anti-Shine-Dalgarno pair permits translation by the mutant 16S rRNA of the selectable marker GFP. 
     
     
         19 . A cell comprising the plasmid of  claim 14 . 
     
     
         20 . The cell of  claim 19 , wherein the mutation in the 16S rRNA gene affects the quantity of selectable marker produced. 
     
     
         21 . The cell of  claim 19 , wherein the cell is a bacterial cell. 
     
     
         22 . The plasmid of  claim 14 , wherein the DNA sequence encoding the 16S rRNA gene is under the control of an inducible promoter.