Nucleic acid fragments for the identification of dechlorinating bacteria

Abstract

A unique 16S rRNA profile derived from Dehalococcoides ethenogenes has been identified and isolated. The profile contains several nucleic acid fragments that are linked to dechlorinating activity. These sequences are set forth in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:30, and SEQ ID NO:34.

1 . An isolated 16S rDNA sequence indicative of a dechlorinating bacterial organism selected from the group consisting of: 
 (a) SEQ ID NO:94 and SEQ ID NO:95    (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS at 65° C.; or an isolated nucleic acid molecule that is completely complementary to (a) or (b).    
     
     
         2 . An isolated 16S rDNA sequence indicative of a dechlorinating bacterial organism selected from the group consisting of: 
 (a) SEQ ID NOs:61-75 and SEQ ID NO:'s 77-93;    (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: O.1X SSC, 0.1% SDS at 65° C; or an isolated nucleic acid molecule that is completely complementary to (a) or (b).    
     
     
         3 . An isolated bacterial organism comprising the isolated 16s rDNA of any of  claim 1  or  claim 2  wherein said organism has the ability to dechlorinate chlorinated compounds.  
     
     
         4 . A method for identifying a dechlorinating bacterial organism comprising: 
 (i) extracting genomic DNA from a bacterial cell suspected of being able to dechlorinate chlorinated compounds;    (ii) probing the extracted genomic DNA with a probe derived from the isolated 16S rDNA sequence of any of claims  1  or  2  under suitable hybridization conditions;    wherein the identification of a hybridizable nucleic acid fragment confirms the presence of a bacteria capable of dechlorinating chlorinated compounds.    
     
     
         5 . A method according to  claim 4  wherein the bacterial cell is a member of a cell population or consortium.  
     
     
         6 . A method according to  claim 4  wherein the genomic DNA is extracted from a cell contained within a soil, groundwater, or microcosm sample.  
     
     
         7 . A method according to  claim 4  wherein the probe corresponds to a portion of any one of the sequences selected from the group consisting of SEQ ID NOs:61-75 and SEQ ID NO's 77-95.  
     
     
         8 . A method according to  claim 4  wherein the probe contains at replication inhibiting moiety at the 3′ end.  
     
     
         9 . A method of  claim 8  wherein the replication inhibiting moiety is selected from the group consisting of dideoxynuleotides, a sequence of mismatched nucleotides, 3′ phosphate, a molecular spacer, and 3′ deoxynucleotides.  
     
     
         10 . The method of  claim 9  where in the 3′ deoxynucleotide is cordycepin.  
     
     
         11 . A method for identifying a dechlorinating bacterial organism comprising: 
 (i) extracting genomic DNA from a cell suspected of being able to dechlorinate chlorinated compounds; and    (ii) amplifying the extracted genomic DNA with at least one oligonucleotide primer corresponding to a portion of the isolated 16s rDNA of any of claims  1  or  2  such that amplification products are generated;    wherein the presence of amplification products confirms the identification of a dechlorinating bacterial organism.    
     
     
         12 . A method according to  claim 11  wherein the oligonucleotide primer corresponds to a portion of any one of the sequences selected from the group consisting of SEQ ID NOs:61-75 and SEQ ID NO's 77-95.  
     
     
         13 . A method according to  claim 11  wherein the genomic DNA is extracted from a cell contained within a soil, groundwater, or microcosm sample.  
     
     
         14 . A method for identifying a dechlorinating bacterial organism comprising: 
 (i) extracting total cellular rRNA from a cell suspected of being able to dechlorinate chlorinated compounds;    (ii) synthesizing complementary DNA strands to the extracted rRNA using a reverse transcriptase and at least one oligonucleotide primer corresponding to a portion of the isolated 16S rDNA sequence of any one of claims  1  or  2 ;    (iii) amplifying the newly generated complementary DNA strands to the extracted rRNA using at least one oligonucleotide primer corresponding to a portion of the isolated 16S rDNA sequence of any one of claims  1  or  2  such that amplification products are generated;    wherein the presence of amplification products confirms the identification of a dechlorinating bacterial organism.    
     
     
         15 . A method according to either of claims  11  or  14  wherein the isolated 16S rDNA sequence of any one of claims  1  or  2  is selected from the group consisting of SEQ ID NO: 79 and SEQ ID NO: 80, and the dechlorinating bacterial organism identified is  Dehalobacter restrictus.    
     
     
         16 . An isolated  Dehalobacter restrictus  identified by the method of  claim 15 .  
     
     
         17 . A method according to either of claims  11  or  14  wherein the isolated 16S rDNA sequence of any one of claims  1  or  2  is selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 82, and the dechlorinating bacterial organism identified is of  Dehalospirillus multivorans.    
     
     
         18 . An isolated  Dehalospirillus multivorans  identified by the method of  claim 17 .  
     
     
         19 . A method according to either of claims  11  or  14  wherein the isolated 16S rDNA sequence of any one of claims  1  or 2 is selected from the group consisting of SEQ ID NO 83, SEQ ID NO: 84, SEQ ID NO:85 and SEQ ID NO:86, and the dechlorinating bacterial organism identified is of  Desulfitobacterium dehalogenans.    
     
     
         20 . An isolated  Desulfitobacterium dehalogenans  identified by the method of  claim 19 .  
     
     
         21 . A method according to either of claims  11  or  14  wherein the isolated 16S rDNA sequence of any one of claims  1  or  2  is selected from the group consisting of SEQ ID NO: 87 or SEQ ID NO:88., and the dechlorinating bacterial organism identified is of  Desulfuromonas chloroethnica.    
     
     
         22 . An isolated  Desulfuromonas chloroethnica  identified by the method of  claim 21 .  
     
     
         23 . A method according to either of claims  1  or  14  wherein the isolated 16S rDNA sequence of any one of claims  1  or  2  is selected from the group consisting of SEQ ID NOs: 89-95, and the dechlorinating bacterial organism identified is of  Dehalococcoides  Family A Group.  
     
     
         24 . An isolated  Dehalococcoides  Family A Group identified by the method of  claim 23 .  
     
     
         25 . A method for the dechlorination of chlorinated compounds comprising contacting a chlorinated compound with the isolated bacterial organism of any one of claims  3 ,  16 ,  18 ,  20 ,  22 , and  24  under conditions for the dechlorination to occur.  
     
     
         26 . A method according to  claim 25  wherein said dechlorinating compound is selected from the group consisting of carbontetrachloride, tetrachloroethene, chloroform, dichloromethane, trichloroethene, dichloroethylene, vinyl chloride, and chloroaromatics, dichloropropane, and chlorinated ethane.  
     
     
         27 . A diagnostic nucleic acid gene fusion useful in Denaturing Gradient Gel Electrophoresis having the general structure: DS-GC, wherein: 
 (i) SS is a signature sequence selected from the group consisting of SEQ ID NOs: 9-29, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO:94, SEQ ID NO:95,    SEQ ID NOs: 35-75, and SEQ ID NOs: 77-93; and    (ii) GC is a GC clamp sequence having the sequence as set forth in SEQ ID NO:76.    
     
     
         28 . The diagnostic nucleic acid fusion of  claim 27  selected from the group consisting of SEQ ID NO:77 and SEQ ID NO:78.  
     
     
         29 . The diagnostic nucleic acid fusion of  claim 27  wherein the GC clamp sequence is attached at either 5′ end of the signature sequence.  
     
     
         30 . A method for separating sub-families of dechlorinating bacterial organisms comprising: 
 (i) extracting total cellular rRNA from a cell suspected of being able to dechlorinate chlorinated compounds;    (ii) synthesizing complementary DNA strands to the extracted rRNA using a reverse transcriptase and at least one oligonucleotide primer corresponding to a portion of the diagnostic gene fusion of  claim 27  such that amplification products are generated;    (iii) amplifying the newly generated complementary DNA strands to the extracted rRNA of step (ii) using at least one oligonucleotide primer corresponding to a portion of the diagnostic gene fusion of  claim 21  such that amplification products are generated; and    (iv) separating the amplification products by Denaturing Gradient Gel Electrophoresis.    
     
     
         31 . An isolated 16S rDNA sequence indicative of a dechlorinating bacterial strain selected from the group consisting of: 
 (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:8, SEQ ID NO:30, and SEQ ID NO:34;    (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS at 65° C.; and    (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).    
     
     
         32 . An isolated 16S rDNA sequence indicative of a dechlorinating bacterial strain selected from the group consisting of: 
 (a) SEQ ID NOs:9-29 and SEQ ID NOs:35-60;    (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS at 65° C.; and    (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).