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Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

ABSTRACT: The transcription of reductive dehalogenase homologous (rdh) genes of "Dehalococcoides" sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3- and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of the transcription of 29 rdhA genes was indicated in response to 1,2,3- and 1,2,4-TCB added after a substrate depletion period of 72 h. The transcription of the remaining three rdhA genes additionally was detected using specific primers. While most rdhA genes were upregulated similarly in cultures after induction with 1,2,3-TCB or 1,2,4-TCB, three rdhA genes responded differentially to 1,2,3- and 1,2,4-TCB, as revealed by the comparison of t-RFLP profiles. The enhanced transcription of cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TCB, while the transcription of cbdbA1624 was strongly induced by 1,2,4-TCB. Comparison of t-RFLP profiles obtained from cDNA and genomic DNA indicated a particularly high induction of the transcription of cbrA (=cbdbA84) by both TCBs. As indicated by reverse transcription-quantitative PCR, the transcription of these plus two other rdhA genes (cbdbA1588 and cbdbA1618) increased within 48 to 72 h by one or two orders of magnitude. Subsequently, transcript levels slowly decreased and approached initial transcript levels several days after complete dehalogenation. Finally, cbrA was transcribed to a level of 22 transcripts per cbrA gene, suggesting that cbrA mRNA could be an appropriate biomarker for the investigation of the natural dechlorination potential at chlorobenzene-contaminated sites.

SUBMITTER: Wagner A

PROVIDER: S-EPMC2663209 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

Wagner Anke A Adrian Lorenz L Kleinsteuber Sabine S Andreesen Jan R JR Lechner Ute U

Applied and environmental microbiology 20090205 7

The transcription of reductive dehalogenase homologous (rdh) genes of "Dehalococcoides" sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3- and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of t ...[more]

PMID: 19201984

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Project description:Polybrominated diphenyl ethers (PBDEs) are persistent, highly toxic, and widely distributed environmental pollutants. The microbial populations and functional reductive dehalogenases (RDases) responsible for PBDE debromination in anoxic systems remain poorly understood, which confounds bioremediation of PBDE-contaminated sites. Here, we report a PBDE-debrominating enrichment culture dominated by a previously undescribed Dehalococcoides mccartyi population. A D. mccartyi strain, designated TZ50, whose genome contains 25 putative RDase-encoding genes, was isolated from the debrominating enrichment culture. Strain TZ50 dehalogenated a mixture of pentabrominated diphenyl ether (penta-BDE) and tetra-BDE congeners (total BDEs, 1.48 μM) to diphenyl ether within 2 weeks (0.58 μM Br-/day) via ortho- and meta-bromine elimination; strain TZ50 also dechlorinated tetrachloroethene (PCE) to vinyl chloride and ethene (260.2 μM Cl-/day). Results of native PAGE, proteomic profiling, and in vitro enzymatic activity assays implicated the involvement of three RDases in PBDE and PCE dehalogenation. TZ50_0172 (PteATZ50) and TZ50_1083 (TceATZ50) were responsible for the debromination of penta- and tetra-BDEs to di-BDE. TZ50_0172 and TZ50_1083 were also implicated in the dechlorination of PCE to trichloroethene (TCE) and of TCE to vinyl chloride/ethene, respectively. The other expressed RDase, TZ50_0090 (designated BdeA), was associated with the debromination of di-BDE to diphenyl ether, but its role in PCE dechlorination was unclear. Comparatively few RDases are known to be involved in PBDE debromination, and the identification of PteATZ50, TceATZ50, and BdeA provides additional information for evaluating debromination potential at contaminated sites. Moreover, the ability of PteATZ50 and TceATZ50 to dehalogenate both PBDEs and PCE makes strain TZ50 a suitable candidate for the remediation of cocontaminated sites. IMPORTANCE The ubiquity, toxicity, and persistence of polybrominated diphenyl ethers (PBDEs) in the environment have drawn significant public and scientific interest to the need for the remediation of PBDE-contaminated ecosystems. However, the low bioavailability of PBDEs in environmental compartments typically limits bioremediation of PBDEs and has long impeded the study of anaerobic microbial PBDE removal. In the current study, a novel Dehalococcoides mccartyi strain, dubbed strain TZ50, that expresses RDases that mediate organohalide respiration of both PBDEs and chloroethenes was isolated and characterized. Strain TZ50 could potentially be used to remediate multiple cooccurring organohalides in contaminated systems.

Project description:Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

Dataset Information

Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

Publications

Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

Similar Datasets

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