Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities

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Abstract

We developed standard curves based on plasmids containing a 16S rRNA gene of a member of one of the three genera Dehalobacter, Desulfitobacterium, and Dehalococcoides. A large difference in amplification efficiency between the standard curves was observed ranging from 1.5 to 2.0. The total eubacterial 16S rRNA gene copy number determined in a sample DNA by using eubacterial primers and the three standard curves led to differences in the estimated copy numbers of a factor up to 73. However, the amplification efficiencies for one specific standard curve were the same independent of the PCR primer pair used. This allowed the determination of the abundance of a population expressed as fractional number, hence, the percentage of genus-specific copy numbers within the total eubacterial 16S rRNA gene copy numbers. Determination of the fractional numbers in DNA mixtures of known composition showed the accuracy of this approach. The average difference in threshold value between two 10-fold dilutions of DNA of pure cultures, mixtures thereof and of environmental samples was −3.45±0.34, corresponding to an average almost optimal amplification efficiency of 1.95. This indicated that the low amplification efficiency of certain standard curves seemed to be mainly a problem of the plasmid DNA used and not of the 16S rRNA gene of the target genera.

Introduction

Chloroethenes have been entering the environment as a result of improper handling, storage or spillage. Due to their low degradation potential under aerobic conditions, they can seep into groundwater. There, degradation under anaerobic conditions, either by oxidation under iron- or manganese-reducing conditions (Bradley and Chapelle, 1998) or by complete reductive dechlorination to ethene (Holliger et al., 1999), can take place. Reductive dechlorination is considered as the major process for the biodegradation of tetra- (PCE) and trichloroethene (TCE). However, at several sites the reductive dechlorination of PCE and TCE is not complete, yielding the more toxic intermediates dichloroethene (DCE) and vinyl chloride (VC). Most of the chloroethene-dechlorinating isolates, including Dehalobacter restrictus (Holliger et al., 1998), several Desulfitobacterium spp. Bouchard et al., 1996, Gerritse et al., 1999, Suyama et al., 2001 and Sulfurospirillum spp. Scholz-Muramatsu et al., 1995, Luijten et al., 2003, dechlorinate PCE and TCE to DCE. To date, only one isolate, Dehalococcoides ethenogenes strain 195 (Maymó-Gatell et al., 1997), has been shown to degrade PCE completely to ethene, albeit the last step cometabolically (Maymó-Gatell et al., 1999). Another isolate of the same genus, Dehalococcoides sp. strain BAV1, is able to dechlorinate DCE and VC but not PCE and TCE (He et al., 2003).

For site owners and consulting companies, the estimation of the biodegradation potential at a contaminated site is important to decide on the remediation strategy to be followed. In case of chloroethene-contaminated sites, it would be useful to be able to determine the presence and abundance of known chloroethene-degraders. Culture-based techniques, such as most-probable-number (MPN) determination, are labor-intensive, time-consuming and success is not guaranteed. Culture-independent methods based on molecular biology techniques are less time-consuming alternatives.

Primers for the 16S rRNA gene have been developed for several bacterial genera containing species with dechlorinating properties. A specific primer set has been used to detect Desulfitobacterium spp. in environmental samples from the Canadian province of Quebec Lévesque et al., 1997, Lanthier et al., 2001, whereas primer sets for nested PCR were developed for the detection of Desulfuromonas and Dehalococcoides spp. Löffler et al., 2000, Lendvay et al., 2003. Though highly specific, the detection by either direct or nested PCR can only be used as a qualitative method. For a quantitative detection of Dehalococcoides spp., a competitive PCR method has been developed, but the detection range is relatively narrow (Cupples et al., 2003). PCR-independent methods like ELISA (Bauer-Kreisel et al., 1996) or FISH (Yang and Zeyer, 2003) have also been developed for the quantification of Sulfurospirillum multivorans and Dehalococcoides spp., respectively, but both methods have not been used on environmental samples. For all of the above methods, the largest disadvantage is the relative high detection limit, which does not allow quantification of small populations.

Real-time PCR has been described to be a reliable quantification method. After the introduction of real-time PCR in medical sciences (for recent reviews: see Bustin, 2000, Bustin, 2002, Mackay et al., 2002), the technique has emerged as a mainstream method (Ginzinger, 2002), which has also gained popularity within molecular ecology (e.g. Suzuki et al., 2000, Hermansson and Lindgren, 2001, Stults et al., 2001, Nadkarni et al., 2002, Harms et al., 2003). A great advantage of real-time PCR is the high sensitivity combined with the ability to get quantitative data. We set up real-time PCR assays for three bacterial genera commonly used in our laboratory (D. restrictus strain PER-K23, Desulfitobacterium hafniense strain TCE1 (Gerritse et al., 1999) and D. ethenogenes strain 195). For that purpose, we constructed standard curves from plasmids containing the 16S rRNA gene of each of the target species. While testing, we noticed large differences between the 16S rRNA gene copy numbers for total eubacterial population determined for the same samples when they were quantified via each of the standard curves. Here, we evaluate the differences and give recommendations to circumvent the observed problems.

Section snippets

Strains and chromosomal DNA

Chromosomal DNA of Escherichia coli TOP-10 (Invitrogen, Leek, Groningen) grown on Luria-Bertani (LB) broth was isolated as described before (Sambrook et al., 1989). Chromosomal DNA of D. restrictus PER-K23 and D. hafniense TCE1 was extracted from stationary phase cultures as described by Maillard et al. (2003). Chromosomal DNA of D. ethenogenes strain 195 was obtained from S. Zinder (Cornell University, Ithaka, NY, USA). Chromosomal DNAs of environmental samples and enrichment cultures was

Cloning and sequencing of the D. restrictus strain PER-K23, D. ethenogenes strain 195 and D. hafniense strain TCE1 16S rRNA genes

To complete the sequence of the 16S rRNA gene of D. restrictus strain PER-K23 and for its use as a standard in real-time PCR, the 16S rRNA gene was cloned and 8 clones of the resulting plasmid, pDre1, were sequenced. A 1525-base pair sequence (with primer regions) was obtained, which included the previously determined partial sequence (Holliger et al., 1998) with a few sequence errors corrected. Due to these corrections, the 16S rRNA gene of PER-K23 over the full-length sequence has 99.4%

Discussion

Real-time PCR is considered to be a quantitative PCR method able to give an absolute number for the target sequence in the sample measured. The results in the present study show that giving an absolute number of bacterial 16S rRNA gene copies was not possible due to the variation of the amplification efficiency of the standard curves. Total eubacterial 16S rRNA gene copy numbers can be overestimated over 100-fold depending on the standard curve used and the magnitude of the initial copy number

Acknowledgements

The authors wish to thank Muriel Gaillard for cloning and sequencing of pDco1. We also thank Prof. Stephen H. Zinder (Cornell University, Ithaka, NY, USA) for the chromosomal DNA of D. ethenogenes strain 195 and Dr. Ute Lechner (Martin-Luther-Universität Halle-Wittenberg) for communication of data prior to publication. This research was supported by the Swiss Federal Office for Education and Science (project 01.0119) within the frame of the EC Environment/Water Program CORONA (project

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