16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species

Abstract

Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers.

FIG. 1

Alignment of parts of Desulfuromonas sp. strain BB1's 16S rRNA gene sequence with corresponding regions of phylogenetically related Desulfuromonas species. The sequences shown stem from variable regions and were used to generate Desulfuromonas dechlorinator-targeted oligonucleotide primers. A hyphen indicates a gap, and a dot indicates sequence identity to the 16S rDNA sequence of Desulfuromonas sp. strain BB1.

FIG. 2

Primer specificity. Cell lysates of phylogenetically related Desulfuromonas species were used as templates for the Desulfuromonas dechlorinator-targeted primer pair. Lanes: 1, 1-kb ladder marker; 2, BB1 genomic DNA (positive control); 3, D. acetoxidans; 4, D. thiophila; 5, D. succinoxidans; 6, Desulfuromonas sp. strain BB1; 7, D. chloroethenica; 8, D. acetexigens; 9, no target DNA (negative control).

FIG. 3

Detection of PCE dechlorinators in environmental samples. Total DNA was isolated from 0.25 g of aquifer or sediment material and used as template DNA in a first-round amplification with universal bacterial-specific primers. The Desulfuromonas dechlorinator- and Dehalococcoides-targeted primers were then used in a second amplification phase (nested PCR approach). (A) Nested PCR with the Desulfuromonas dechlorinator-targeted primer pair. (B) Nested PCR with the Dehalococcoides-targeted primer pair. Lanes 1, 1-kb ladder markers; lanes 2 to 6, Bachman aquifer samples 1Bb, 1At, 2Bb, 2At, and 4A, respectively; lanes 7 to 12, aquifer samples from Cape Canaveral, Jacksonville MW510 and IW001, B&J Industrial site C and D, Schoolcraft, respectively; lanes 13, sandy aquifer material used in E. coli experiment; lanes 14 to 19, freshwater sediment samples from Red Cedar River (collected July 1995, May 1998, and November 1998), Père Marquette River (collected April 1995 and September 1998), and Au Sable River, respectively; lanes 20, negative controls.

FIG. 4

RFLP analysis of amplicons obtained with the Desulfuromonas dechlorinator-targeted primers. Amplicons were digested with the restriction endonuclease EcoRI, and fragments were separated on a 2% metaphor agarose gel. Lane 1, DNA marker V; lanes 2 to 9, digested amplicons obtained from Desulfuromonas sp. strain BB1, D. chloroethenica, the Red Cedar River (collected July 1995, May 1998, and November 1998), the Père Marquette River (locations PM 0 and PM 1, collected September 1998), and the Au Sable River, respectively; lane 10, undigested PCR product obtained from D. chloroethenica.