Uropathogenic E. coli D-serine detoxification operon

Abstract

Disclosed are methods of detecting uropathogenic E. coli genes that are differentially expressed in response to D-serine. Also disclosed are methods of characterizing bacterial isolates from clinical samples based on the ability to metabolize D-serine.

We claim:  
     
         1 . A method of detecting uropathogenic  E. coli  nucleotide sequences differentially expressed in the presence or absence of D-serine comprising: 
 (a) providing a library comprising a plurality of transposon mutants of a uropathogenic  E. coli  strain, the mutants comprising a transcriptional fusion comprising a transcriptional regulation sequence operably connected to a sequence encoding a detectable protein;    (b) growing the mutants of (a) in the presence of D-serine;    (c) growing the mutants of (a) in the absence of D-serine;    (d) identifying mutants having increased or decreased expression of the transcriptional fusion in the presence or absence of D-serine by comparing the relative levels of the detectable protein of the mutants of (b) and (c);    (e) identifying the insertion site of the transcriptional fusion in the transposon mutants identified in (d); and    (f) correlating the insertion site with an  E. coli  nucleotide sequence.    
     
     
         2 . The method of  claim 1 , wherein the uropathogenic  E. coli  strain is CFf073.  
     
     
         3 . A method of identifying proteins differentially expressed in a wild-type uropathogenic  E. coli  strain and a uropathogenic  E. coli  dsdCXA locus mutant, the mutant having reduced expression of one or more proteins selected from the group consisting of DsdA, DsdC, and DsdX, the method comprising 
 (a) comparing proteins isolated from the wild-type uropathogenic  E. coli  strain and proteins isolated from the uropathogenic  E. coli  dsdCXA locus mutant; and    (b) identifying proteins from the dsdCXA mutant having increased or decreased level of expression relative to expression of the corresponding proteins in the wild-type uropathogenic  E. coli  strain.    
     
     
         4 . The method of  claim 3 , wherein the mutant is a dsdA mutant.  
     
     
         5 . The method of  claim 3 , wherein the isolated proteins of (a) are separated by two-dimensional gel electrophoresis.  
     
     
         6 . The method of  claim 3 , wherein prior to (a), the wild-type strain and the dsdA mutant are grown in the presence of D-serine.  
     
     
         7 . A method of detecting genes from a uropathogenic  E. coli  strain that are differentially expressed in the presence or absence of D-serine comprising 
 (a) hybridizing a first set of labeled oligonucleotide probes with an array of oligomers, the oligomers comprising gene sequences from the uropathogenic  E. coli , wherein the first set of labeled oligonucleotide probes is made by reverse transcription of RNA isolated from uropathogenic  E. coli  grown in the presence of D-serine;    (b) hybridizing a second set of labeled oligonucleotide probes with an array of oligomers identical to the array of (a), wherein the second set of labeled oligonucleotide probes is made by reverse transcription of RNA isolated from uropathogenic  E. coli  grown in the absence of D-serine;    (c) comparing hybridization of labeled oligonucleotide probes of (a) and (b) to identify oligomers having differential hybridization to the first and second sets of oligonucleotide probes;    (d) identifying genes comprising the sequences of the oligomers of (c).    
     
     
         8 . A method of detecting proteins differentially expressed in a uropathogenic  E. coli  strain in response to D-serine, comprising: 
 (a) providing a first culture of the uropathogenic  E. coli  strain grown in the presence of D-serine;    (b) providing a second culture of the uropathogenic  E. coli  strain grown in the absence of D-serine;    (c) comparing proteins isolated from (a) and (b); and    (d) identifying proteins from  E. coli  grown in the presence of D-serine that are increased or decreased relative to the corresponding proteins in the uropathogenic  E. coli  strain grown in the absence of D-serine.    
     
     
         9 . A method of identifying a uropathogenic  E. coli  polynucleotide sequence that binds to DsdC protein comprising 
 contacting an  E. coli  polynucleotide sequence with DsdC protein; and    detecting binding of the polynucleotide sequence to the protein.    
     
     
         10 . A method of characterizing an  E. coli  strain isolated from a clinical sample comprising testing the strain for the ability to grow in the presence of D-serine.  
     
     
         11 . The method of  claim 10 , wherein D-serine is the sole source of carbon and nitrogen.  
     
     
         12 . The method of  claim 10 , wherein D-serine is present in a concentration effective to inhibit the growth of a normal fecal isolate of  E. coli.    
     
     
         13 . The method of  claim 10 , wherein D-serine is present in a concentration of at least 100 ug/ml.  
     
     
         14 . The method of  claim 10 , wherein D-serine is present at a concentration of from about 100 ug/ml to about 500 ug/ml.  
     
     
         15 . A method of detecting D-serine in a sample comprising the steps of: 
 (a) contacting the sample with a polypeptide comprising D-serine deaminase under suitable reaction conditions and for a period of time sufficient to allow the deamination of at least a portion of D-serine molecules in the sample; and    (b) detecting the deamination of D-serine.    
     
     
         16 . The method of  claim 15 , wherein the polypeptide of step (a) is immobilized on a solid support.  
     
     
         17 . The method of  claim 16 , wherein the polypeptide is coimmobilized with an indicator responsive to ammonia.  
     
     
         18 . A urine dipstick comprising a solid support, a polypeptide comprising D-serine deaminase and an indicator responsive to ammonia, wherein the polypeptide and indicator are coimmobilized on the support.