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.

FIG. 1.

Amino acid alignment of cld gene products from D. agitata (D.agit), I. dechloratans (I.dech), M. magnetotacticum (M.magn), “D. aromatica” (D.arom), Dechloromonas sp. strain LT1 (DcmLT1), Pseudomonas sp. strain PK (Ps. PK), “D. chlorophilus” (D.chlo), D. suillum (D.suil), “D. anomalous” (D.anom), Dechlorospirillum sp. strain DB (st. DB), and strain CR (st. CR). Numbers correspond to residues from the D. agitata mature protein. “∼” denotes alignment gaps; “-” denotes unknown sequence data; “.” denotes identical residues. The DCD-F/DCD-R primer set targeted the lightly shaded regions, while the UCD-238F/UCD-646 primer set targeted the darkly shaded regions.

FIG. 2.

Amplification of a 484-bp internal region of the cld gene using the DCD-F/DCD-R primer set. Lane 1, “D. aromatica”; lane 2, M. magnetotacticum; lane 3, I. dechloratans; lane 4, Pseudomonas sp. strain PK; lane 5, D. suillum; lane 6, “D. chlorophilus”; lane 7, “D. anomalous”; lane 8, D. strain LT1; lane 9, negative control (no DNA); lane 10, 1-kb ladder.

FIG. 3.

Amplification of a 408-bp internal region of the cld gene using the UCD-238F/UCD-646R primer set. Lane 1, D. agitata; lane 2, “D. aromatica”; lane 3, M. magnetotacticum; lane 4, I. dechloratans; lane 5, Pseudomonas sp. strain PK; lane 6, D. suillum; lane 7, “D. chlorophilus”; lane 8, “D. anomalous”; lane 9, Dechloromonas species strain LT-1; lane 10, R. tenuis; lane 11, P. stutzeri; lane 12, Escherichia coli; lane 13, negative control (no DNA); lane 14, 100-bp ladder.

FIG. 4.

Testing of the universal cld gene primer sets on environmental DNAs. Top of gel, touchdown PCR using the DCD-F/DCD-R primer set, corresponding to a 484-bp internal region of the cld gene. Bottom of gel, nested PCR on the above reactions, using the UCD-238F/UCD-646R primer set, corresponding to a 408-bp internal region of the cld gene. Lane 1, Pseudomonas sp. strain PK (positive control); lane 2, Los Alamos well 2; lane 3, Los Alamos well 3; lane 4, Los Alamos well 4; lane 5, Los Alamos well 5; lane 6, Los Alamos well 7; lane 7, campus library pond; lane 8, campus library soil; lane 9, campus lake; lane 10, Lake Fryxell sediment; lane 11, Lake Fryxell 7-m water column; lane 12, Lake Fryxell 12-m water column; lane 13, Lake Hoare 12-m water column; lane 14, Lake Hoare mat; lane 15, Vida; lane 16, negative control (no DNA); lane 17, 100-bp ladder.

FIG. 5.

cld and 16S rDNA phylogenetic trees. (A) cld phylogenetic tree generated from an alignment of 369 bp. (B) 16S rDNA phylogenetic tree generated from an alignment of 1,424 bp. The numbers correspond to bootstrap values from 100 replicates. D. agitata (Dcm.agit), “D. aromatica” (Dcm.arom), D. strain LT1 (Dcm.LT1), D. suillum (Dcs. suil), “D. anomalous” (Dsp.anom), “D. chlorophilus” (Dma.chlo), I. dechloratans (I.dech), M. magnetotacticum (M.magn), Pseudomonas sp. strain PK (Pseud.PK), Dechlorospirillum sp. strain DB (DB), and strain CR (CR) were analyzed.