Materials and methods for detection of Oxalobacter formigenes

Abstract

The subject invention concerns the novel use of formyl-CoA transferase enzyme together with oxalyl-CoA decarboxylase enzyme for the detection and measurement of oxalate in biological samples. The use of the enzyme system according to the subject invention results in the conversion of oxalate into carbon dioxide and formate. Because the production of formate is directly correlated to the concentration of oxalate present in a sample, the determination of the resulting formate concentration provides an accurate, sensitive and rapid means for detecting even low levels of oxalate. The subject invention further concerns the cloning, sequencing and expression of the genes that encode the formyl-CoA transferase enzyme and the oxalyl-CoA decarboxylase enzyme of Oxalobacter formigenes. The subject invention also concerns methods for detecting the presence of Oxalobacter formigenes organisms in a sample, and the polynucleotide probes and primers used in the detection method.

We claim:  
     
         1 . A purified polynucleotide molecule, comprising a nucleotide sequence that encodes an oxalyl-CoA decarboxylase polypeptide, or a fragment of said oxalyl-CoA decarboxylase that retains functional enzymatic activity.  
     
     
         2 . The polynucleotide molecule according to  claim 1 , wherein said nucleotide sequence is derived from  Oxalobacter formigenes.    
     
     
         3 . The polynucleotide molecule according to  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, or a fragment of said oxalyl-CoA decarboxylase that retains functional enzymatic activity.  
     
     
         4 . The polynucleotide molecule according to  claim 1 , comprising the nucleotide sequence shown in SEQ ID NO. 3.  
     
     
         5 . The polynucleotide molecule according to  claim 1 , wherein said polynucleotide molecule hybridizes under standard high-stringency conditions with a polynucleotide molecule comprising the nucleotide sequence shown in SEQ ID NO. 3, or the complementary sequence thereof.  
     
     
         6 . The polynucleotide molecule according to  claim 1 , wherein said polynucleotide consists of a nucleotide sequence that encodes oxalyl-CoA decarboxylase comprising the amino acid sequence shown in SEQ ID NO. 4, or a fragment of said oxalyl-CoA decarboxylase that retains functional enzymatic activity.  
     
     
         7 . The polynucleotide molecule according to  claim 6 , wherein said polynucleotide hybridizes under high stringency conditions with a nucleotide sequence comprising nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.  
     
     
         8 . The polynucleotide according to  claim 6 , wherein said nucleotide sequence consists of nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.  
     
     
         9 . A polynucleotide probe, comprising a nucleotide sequence that is substantially complementary with a polynucleotide sequence present in an  Oxalobacter formigenes  genome, wherein the polynucleotide sequence present in the  Oxalobacter formigenes  genome comprises a gene selected from the group consisting of the formyl-CoA transferase gene and the oxalyl-CoA decarboxylase gene.  
     
     
         10 . A polynucleotide PCR primer, comprising a nucleotide sequence that is substantially complementary with a polynucleotide sequence present in an  Oxalobacter formigenes  genome, wherein said polynucleotide sequence present in said  Oxalobacter formigenes  genome comprises a gene selected from the group consisting of the formyl-CoA transferase gene and the oxalyl-CoA decarboxylase gene, and wherein said PCR primer is capable of priming PCR amplification of said polynucleotide sequence present in said  Oxalobacter formigenes  genome.  
     
     
         11 . A method for detecting  Oxalobacter formigenes  in a sample, comprising the steps of: 
 (a) contacting said sample with a polynucleotide probe according to  claim 9  under conditions sufficient for selective hybridization of said polynucleotide probe with a DNA fragment specific for  Oxalobacter formigenes;  and    (b) detecting said probe hybridized to said DNA fragment.    
     
     
         12 . A polynucleotide vector comprising a polynucleotide molecule according to  claim 1 .  
     
     
         13 . The polynucleotide vector according to  claim 12 , wherein said polynucleotide molecule consists of a nucleotide sequence that encodes an oxalyl-CoA decarboxylase polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, or a fragment of said oxalyl-CoA decarboxylase that retains functional enzymatic activity.  
     
     
         14 . The polynucleotide vector according to  claim 12 , wherein said polynucleotide vector hybridizes under high stringency conditions with a nucleotide sequence consisting of nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.  
     
     
         15 . The polynucleotide vector according to  claim 12 , wherein said nucleotide sequence of said polynucleotide molecule consists of nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.  
     
     
         16 . A recombinant host cell which comprises the polynucleotide vector of  claim 12 , and wherein said cell expresses said oxalyl-CoA decarboxylase or a fragment of said oxalyl-CoA decarboxylase that retains enzymatic activity.  
     
     
         17 . The cell according to  claim 16 , wherein said polynucleotide vector hybridizes under high stringency conditions with a nucleotide sequence consisting of nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.  
     
     
         18 . The cell according to  claim 16 , wherein said nucleotide sequence of said polynucleotide molecule consists of nucleotides 181 through 1884 of the nucleotide sequence shown in SEQ ID NO. 3 or the complementary sequence thereof.