Metabolic Primers for Detection of (Per)chlorate-Reducing Bacteria in the Environment and Phylogenetic Analysis of cld Gene Sequences

Abstract

Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.

Biology of Pseudomonas stutzeri

“…A recent comparison of a phylogenetic tree based on 16S rRNA with a tree developed from the cld gene sequences of 11 diverse DPRB demonstrated significant discrepancies. The results of this comparison supported evolution through horizontal gene transfer (22). As mentioned above, strain AW1 is a member of genomovar 3.…”

Genome Sequences of Alicycliphilus denitrificans Strains BC and K601 T

“…This finding explains the predicted horizontal gene transfer of genes involved in chlorate respiration (1). Furthermore, genes coding for a benzene/phenol monooxygenase (BC-BMOa) and a catechol 2,3-dioxygenase (BC-C23O), both postulated to be involved in benzene degradation (8), are localized in one gene cluster in the chromosomes of strain BC and strain K601…”

Unexpected Diversity of Chlorite Dismutases: a Catalytically Efficient Dimeric Enzyme from Nitrobacter winogradskyi

“…The presence of this gene on a mobile genetic element indicates that lineage II cld genes are subject to horizontal transfer and may be widespread among environmental bacteria. Existing PCR primers, which target the canonical lineage I cld genes (3,33) and were used to detect these genes in the environment (3) and in pure cultures (6), have numerous base mismatches to all lineage II genes (data not shown). Thus, the environmental distribution of lineage II cld genes, for example at (per)chlorate-contaminated sites, is unknown at present.…”