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 . A method as in  claim 1  wherein said assay comprises 
 a determination of the levels of a transcription product of said genetic locus.  
 
     
     
         3 . A method as in  claim 1  wherein said assay comprises 
 a determination of the levels of a translation product of said genetic locus.  
 
     
     
         4 . A method as in  claim 1  further comprising 
 introducing within said bacteria a nucleotide sequence, said sequence including 
 at least a functional fragment of a regulatory region of a bacterial multiple antibiotic resistance operon operably joined to a marker locus such that the expression of said marker locus is substantially dependent upon said fragment of said regulatory region, and  
 wherein said genetic locus is said marker locus and said assay is for the expression of said marker locus.  
 
 
     
     
         5 . A method as in  claim 4  wherein said marker locus is a sequence encoding an enzyme.  
     
     
         6 . A method as in  claim 5  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial beta-galactosidase.  
     
     
         7 . A method as in  claim 4  further comprising 
 introducing within said bacteria an operable nucleotide sequence encoding at least a functional fragment of a repressor of said bacterial multiple antibiotic resistance operon,  
 wherein said fragment of said repressor is capable of substantially decreasing the expression of a bacterial multiple antibiotic resistance operon, and  
 wherein said assay is for increased expression of said genetic locus.  
 
     
     
         8 . A method as in  claim 4  wherein, prior to said exposure, said bacteria effectively express a multiple antibiotic resistance phenotype and 
 wherein said assay is for decreased expression of said genetic locus.  
 
     
     
         9 . A method of predicting the antibiotic effectiveness of a composition comprising 
 exposing a strain of bacteria to said composition and    determining the effects of said composition on the growth of said bacteria    wherein, prior to said exposure, said strain of bacteria has been treated so as to substantially decrease expression of a multiple antibiotic resistance operon.    
     
     
         10 . A method as in  claim 9  wherein said operon has been at least partially deleted in said bacteria such that the expression of an activator locus of said operon is substantially decreased.  
     
     
         11 . A method as in  claim 9  wherein said strain of bacteria has been genetically altered, said alteration comprising 
 introducing within said bacteria an operable nucleotide sequence encoding at least a functional fragment of a repressor of said operon  
 wherein said fragment of said repressor is capable of substantially decreasing the expression of said operon.  
 
     
     
         12 . A method as in  claim 9  wherein said strain of bacteria has been genetically altered, said alteration comprising 
 introducing within said bacteria an operable nucleotide sequence encoding an mRNA transcript characterized by substantial homology to a least a fragment of an activator locus of said operon such that said mRNA transcript substantially decreases the expression of said activator locus.  
 
     
     
         13 . A method as in  claim 9  wherein said strain of bacteria has been genetically altered, said alteration comprising 
 introducing within said bacteria a transposon such that said transposon inserts within an activator locus of said operon.  
 
     
     
         14 . A method of predicting the antibiotic effectiveness of a composition comprising 
 exposing a strain of bacteria to said composition and    determining the effects of said composition on said bacteria    wherein, prior to said exposure, said strain of bacteria has been treated so as to substantially increase the expression of a bacterial multiple antibiotic resistance operon.    
     
     
         15 . A method as in  claim 14  wherein said operon has been at least partially deleted in said bacteria.  
     
     
         16 . A method as in  claim 14  wherein said strain of bacteria has been genetically altered, said alteration comprising 
 introducing within said bacteria an operable nucleotide sequence encoding at least a functional fragment of an activator of said operon  
 wherein said fragment is capable of substantially increasing the expression of a bacterial multiple antibiotic resistance phenotype.  
 
     
     
         17 . A method as in  claim 14  wherein said strain of bacteria has been genetically altered, said alteration comprising 
 introducing within said bacteria an operable nucleotide sequence encoding an mRNA transcript that is characterized by substantial homology to a least a fragment of a repressor locus of said operon such that said mRNA transcript substantially decreases the expression of said repressor locus.  
 
     
     
         18 . A method as in  claim 1  further comprising 
 introducing within said bacteria a nucleotide sequence, said sequence including 
 a marker locus operably joined to at least a functional fragment of a regulatory region of a bacterial operon such that the expression of said marker locus is substantially dependent upon said fragment of a regulatory region of said bacterial operon,  
 wherein the expression of said bacterial operon is regulated at least in part by expression of a bacterial multiple antibiotic resistance operon.  
 
 
     
     
         19 . A method as in  claim 18  wherein said bacteria express a multiple antibiotic resistance phenotype.  
     
     
         20 . A method as in  claim 18  wherein a repressor locus of said operon has been at least partially deleted such that said bacteria express a multiple antibiotic resistance phenotype.  
     
     
         21 . A method as in  claim 18  further comprising 
 introducing within said bacteria an operable nucleotide sequence encoding at least a functional fragment of an activator of said bacterial multiple antibiotic resistance operon  
 wherein said fragment is capable of substantially increasing the expression of a bacterial multiple antibiotic resistance phenotype.  
 
     
     
         22 . A method as in  claim 21  wherein a repressor locus of said operon has been at least partially deleted such that said bacteria express a multiple antibiotic resistance phenotype.  
     
     
         23 . A method as in  claim 18  further comprising 
 introducing within said bacteria an operable nucleotide sequence encoding an mRNA transcript characterized by substantial homology to at least a fragment of a repressor locus of said operon such that said mRNA transcript substantially decreases the expression of said repressor locus.  
 
     
     
         24 . A method as in  claim 22  wherein said marker locus encodes at least a functional fragment of a bacterial beta-galactosidase and said bacterial operon is a bacterial micF operon.  
     
     
         25 . A method of inhibiting the growth of bacteria comprising 
 exposing said bacteria to a composition including an amount of an antibiotic composition and an amount of a substance which substantially decreases the expression of a multiple antibiotic resistance phenotype by said bacteria.    
     
     
         26 . A method as in  claim 25  wherein said substance is a nucleotide sequence comprising at least a functional fragment of a repressor locus of a bacterial multiple antibiotic resistance operon operably joined to a regulatory region such that the expression of said repressor locus is substantially dependent upon said regulatory region and 
 said sequence is free from operable sequences encoding an activator of a bacterial multiple antibiotic resistance operon.  
 
     
     
         27 . A method as in  claim 25  wherein said substance is a nucleotide sequence characterized by substantial homology to at least a fragment of an activator locus of a bacterial multiple antibiotic resistance operon.  
     
     
         28 . A method as in  claim 25  wherein said substance is an inhibitor of the activity of an activator of a bacterial multiple antibiotic resistance operon.  
     
     
         29 . A method of identifying bacterial loci which affect resistance to antibiotic compositions comprising 
 allowing an activator of a bacterial multiple antibiotic resistance operon to bind to a bacterial DNA molecule and    assaying for sites on said DNA to which said activator binds.    
     
     
         30 . A method as in  claim 29  wherein said DNA has been fragmented and said assay is in vitro.  
     
     
         31 . A method of identifying bacterial loci which affect resistance to antibiotic compositions comprising 
 introducing within said bacteria an operable nucleotide sequence encoding at least a functional fragment of an activator of a bacterial multiple-antibiotic resistance operon    wherein said fragment of said activator is capable of substantially increasing the expression of a bacterial multiple antibiotic resistance phenotype, and    assaying for changes in the levels of expression of said loci within said bacteria.    
     
     
         32 . A method of identifying bacterial loci which affect resistance to antibiotic compositions comprising 
 subjecting bacteria to a first set of conditions such that said bacteria express a multiple antibiotic resistance phenotype,    introducing within said bacteria a nucleotide sequence including a marker locus and free of a regulatory region operably joined to said marker locus,    permitting said sequence to integrate at random sites within a chromosome of said bacteria,    assaying for expression of said marker locus,    subjecting a subset of said bacteria which express said marker locus to a second set of conditions such that said subset of bacteria do not express said phenotype;    assaying for bacteria in said subset of bacteria which do not express said marker locus under said second set of conditions, and    determining said site of integration of said marker locus in said subset of bacteria which express said marker locus under said first set of conditions and which do not express said marker locus under said second set of conditions.    
     
     
         33 . A method as in  claim 32  wherein said first set of conditions comprises 
 introducing within said bacteria a temperature sensitive plasmid including an operable nucleotide sequence encoding at least a functional fragment of an activator of a bacterial multiple antibiotic resistance operon,  
 wherein said fragment of said activator is capable of substantially increasing the expression of a bacterial multiple antibiotic resistance phenotype,  
 wherein said second set of conditions comprises 
 increasing the temperature under which said bacteria are cultured such that the replication of said temperature sensitive plasmid is substantially inhibited, wherein 
 said bacteria are free of an operable activator locus of a bacterial multiple antibiotic resistance operon on a chromosome of said bacteria, and wherein 
 said bacteria are recombination deficient.  
 
 
 
 
     
     
         34 . A method as in  claim 32  wherein said first set of conditions comprises 
 introducing within said bacteria a temperature sensitive plasmid including an operable nucleotide sequence encoding an mRNA transcript characterized by substantial homology to at least a fragment of a repressor locus of a bacterial multiple antibiotic resistance operon such that said transcript substantially decreases the expression of said repressor locus, and wherein said second set of conditions comprises 
 increasing the temperature under which said bacteria are cultured such that the replication of said temperature sensitive plasmid is substantially inhibited, wherein 
 said bacteria possess an operable bacterial multiple antibiotic resistance operon on a chromosome of said bacteria, and wherein 
 said bacteria are recombination deficient.  
 
 
 
 
     
     
         35 . A method as in  claim 32  wherein said second set of conditions comprises 
 exposing said bacteria to a transposon such that said transposon enters said bacteria and inactivates an activator locus of a bacterial multiple antibiotic resistance operon by insertion within said locus.  
 
     
     
         36 . A method as in  claim 33  wherein said marker locus is a sequence encoding an enzyme.  
     
     
         37 . A method as in  claim 36  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial beta-galactosidase.  
     
     
         38 . A method as in  claim 36  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial alkaline phosphatase.  
     
     
         39 . A method of identifying bacterial loci which affect resistance to antibiotic compositions comprising 
 subjecting bacteria to a first set of conditions such that said bacteria do not express a multiple antibiotic resistance phenotype,    introducing within said bacteria a nucleotide sequence including a marker locus and free of a regulatory region operably joined to said marker locus,    permitting said sequence to integrate within a chromosome of said bacteria,    assaying for expression of said marker locus,    subjecting a subset of said bacteria which do not express said marker locus to a second set of conditions such that said subset of bacteria express said phenotype;    assaying for bacteria in said subset of bacteria which express said marker locus under said second set of conditions, and    determining the site of integration of said marker locus in said subset of bacteria which do not express said marker locus under said first set of conditions and which express said marker locus under said second set of conditions.    
     
     
         40 . A method as in  claim 39  wherein said first set of conditions comprises 
 growing said bacteria in a culture substantially free of inducers of a bacterial multiple antibiotic resistance operon,  
 wherein said second set of conditions comprises 
 exposing said bacteria to an inducer of a bacterial multiple antibiotic resistance operon such that said inducer enters said bacteria and induces expression of a multiple antibiotic resistance phenotype in said bacteria.  
 
 
     
     
         41 . A method as in  claim 40  wherein said marker locus is a sequence encoding an enzyme.  
     
     
         42 . A method as in  claim 41  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial beta-galactosidase.  
     
     
         43 . A method as in  claim 41  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial alkaline phosphatase.  
     
     
         44 . A composition comprising 
 an isolated nucleotide sequence, including 
 at least a functional fragment of a regulatory region of a bacterial multiple antibiotic resistance operon operably joined to a marker locus such that the expression of said marker locus is substantially dependent upon said fragment, wherein said marker locus is a locus other than a bacterial multiple antibiotic resistance operon locus.  
   
     
     
         45 . The composition of  claim 44  wherein said marker locus is a sequence encoding an enzyme.  
     
     
         46 . The composition of  claim 45  wherein said marker locus is a sequence encoding at least a functional fragment of a bacterial beta-galactosidase.  
     
     
         47 . A composition comprising 
 an isolated nucleotide sequence including at least a fragment of a bacterial multiple antibiotic resistance operon.    
     
     
         48 . A composition comprising 
 an isolated nucleotide sequence,    wherein said sequence is sufficiently homologous to a bacterial multiple antibiotic resistance operon so as to be capable of binding in a sequence specific manner to said operon, and    wherein said sequence is of sufficient length such that said binding distinguishes said operon from loci other than bacterial multiple antibiotic resistance operon loci.    
     
     
         49 . The composition of  claim 48  wherein said sequence comprises 
 at least a functional fragment of a repressor locus of a bacterial multiple antibiotic resistance operon operably joined to a regulatory region such that the expression of said repressor is substantially dependent upon said regulatory region, and  
 said sequence is free from operable sequences encoding an activator of a bacterial multiple antibiotic resistance operon.  
 
     
     
         50 . The composition of  claim 48  wherein said sequence comprises 
 an anti-sense locus operably joined to a regulatory region such that the expression of said anti-sense locus is substantially dependent upon said regulatory region,  
 said anti-sense locus encodes an mRNA transcript characterized by substantial anti-sense homology to at least a fragment of a DNA or mRNA of an activator locus of said bacterial multiple antibiotic resistance operon and  
 said sequence is free of operable sequences encoding a repressor locus of said bacterial multiple antibiotic resistance operon.  
 
     
     
         51 . The composition of  claim 48  wherein said sequence comprises 
 at least a functional fragment of an activator locus of a bacterial multiple antibiotic resistance operon operably joined to a regulatory region such that the expression of said activator gene is substantially dependent upon said regulatory region and  
 said sequence is free of operable sequences encoding a repressor of a bacterial multiple antibiotic resistance operon.  
 
     
     
         52 . The composition of  claim 48  wherein said sequence comprises 
 an anti-sense locus operably joined to a regulatory region such that the expression of said anti-sense locus is substantially dependent upon said fragment of a regulatory region,  
 said anti-sense locus encodes an mRNA transcript characterized by substantial homology to at least a fragment of a repressor locus of said bacterial multiple antibiotic resistance operon and  
 said sequence is free of operable sequences encoding an activator of said bacterial multiple antibiotic resistance operon.  
 
     
     
         53 . The composition of  claim 51  wherein said sequence is included in a temperature sensitive plasmid.  
     
     
         54 . The composition of  claim 52  wherein said sequence is included in a temperature sensitive plasmid.  
     
     
         55 . An antibacterial composition comprising 
 an antibiotic composition and    a substance which substantially decreases the expression of a bacterial multiple antibiotic resistance operon.    
     
     
         56 . An antibacterial composition as in  claim 55  wherein the substance is a nucleotide sequence, said sequence comprising 
 at least a functional fragment of a repressor locus of a bacterial multiple antibiotic resistance operon operably joined to a regulatory region such that the expression of said repressor locus is substantially dependent upon said regulatory region and  
 said sequence is free from operable sequences encoding an activator of a bacterial multiple antibiotic resistance operon.  
 
     
     
         57 . An antibacterial composition as in  claim 55  wherein the substance is a nucleotide sequence characterized by substantial homology to at least a fragment of an activator locus of a bacterial multiple antibiotic resistance operon such that said composition is capable of substantially decreasing the expression of said locus.  
     
     
         58 . An antibacterial composition as in  claim 55  wherein said substance is an inhibitor of the activity of an activator of a bacterial multiple antibiotic resistance operon.  
     
     
         59 . A composition comprising 
 substantially pure repressor of a bacterial multiple antibiotic resistance operon.    
     
     
         60 . A composition as in  claim 59  wherein said repressor is labeled.  
     
     
         61 . A composition comprising 
 substantially pure activator of a bacterial multiple antibiotic resistance operon.    
     
     
         62 . A composition as in  claim 61  wherein said activator is labeled.  
     
     
         63 . A composition comprising 
 a substantially pure nucleotide sequence having a sequence characterized by substantial homology to at least a fragment of a repressor locus of a bacterial multiple antibiotic resistance operon    wherein said nucleotide sequence has sufficient homology so as to be capable of binding in a sequence specific manner to said fragment of said repressor locus, and    wherein said nucleotide sequence is of sufficient length such that said binding distinguishes said fragment of said repressor locus from loci other than bacterial multiple antibiotic resistance operon repressor loci.    
     
     
         64 . A composition as in  claim 63  wherein said nucleotide sequence is labeled.  
     
     
         65 . A composition comprising 
 substantially pure nucleotide sequence having a sequence characterized by substantial homology to at least a fragment of an activator locus of a bacterial multiple antibiotic resistance operon    wherein said nucleotide sequence has sufficient homology so as to be capable of binding in a sequence specific manner to said fragment of said activator locus, and    wherein said nucleotide sequence is of sufficient length such that said binding distinguishes said fragment of said activator locus from loci other than bacterial multiple antibiotic resistance operon activator loci.    
     
     
         66 . A composition as in  claim 65  wherein said nucleotide sequence is labeled.  
     
     
         67 . Bacterial cells into which have been introduced a composition selected from the group consisting of the composition of  claim 44 , the composition of  claim 45 , the composition of  claim 46 , the composition of  claim 47 , the composition of  claim 48 , the composition of  claim 49 , the composition of  claim 50 , the composition of  claim 51 , and the composition of  claim 52 .  
     
     
         68 . Bacterial cells into which have been introduced the composition of  claim 53 .  
     
     
         69 . Bacterial cells as in  claim 68  wherein said bacteria are recombination deficient.  
     
     
         70 . Bacterial cells as in any one of claims  68  or  69  wherein said bacteria are free of an operable multiple antibiotic resistance operon on a chromosome.  
     
     
         71 . Bacterial cells into which have been introduced the composition of  claim 54 .  
     
     
         72 . Bacterial cells as in  claim 71  wherein said bacteria are recombination deficient.  
     
     
         73 . An isolated nucleotide sequence comprising SEQ ID NO: 1.  
     
     
         74 . An isolated nucleotide sequence encoding SEQ ID NO: 2.  
     
     
         75 . An isolated nucleotide sequence encoding SEQ ID NO: 3.  
     
     
         76 . An isolated nucleotide sequence encoding SEQ ID NO: 4.  
     
     
         77 . An isolated nucleotide sequence encoding SEQ ID NO: 5.  
     
     
         78 . An isolated nucleotide sequence encoding SEQ ID NO: 6.  
     
     
         79 . An isolated nucleotide sequence encoding SEQ ID NO: 7.  
     
     
         80 . A substantially pure protein corresponding to SEQ ID NO: 2 or a fragment thereof.  
     
     
         81 . A substantially pure protein corresponding to SEQ ID NO: 3 or a fragment thereof.  
     
     
         82 . A substantially pure protein corresponding to SEQ ID NO: 4 or a fragment thereof.  
     
     
         83 . A substantially pure protein corresponding to SEQ ID NO: 5 or a fragment thereof.  
     
     
         84 . A substantially pure protein corresponding to SEQ ID NO: 6 or a fragment thereof.  
     
     
         85 . A substantially pure protein corresponding to SEQ ID NO: 7 or a fragment thereof.