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Applied and Environmental Microbiology, April 2009, p. 1876-1884, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01042-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Transcription Analysis of Genes Encoding Homologues of Reductive Dehalogenases in "Dehalococcoides" sp. Strain CBDB1 by Using Terminal Restriction Fragment Length Polymorphism and Quantitative PCR{triangledown} ,{dagger}

Anke Wagner,1,{ddagger} Lorenz Adrian,2,3 Sabine Kleinsteuber,3 Jan R. Andreesen,1 and Ute Lechner1*

Institut für Biologie/Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle, Germany,1 Fachgebiet Technische Biochemie, Technische Universität 13353 Berlin, Germany,2 Helmholtz Centre for Environmental Research—UFZ, 04318 Leipzig, Germany3

Received 9 May 2008/ Accepted 23 January 2009

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.

* Corresponding author. Mailing address: Institut für Biologie/Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany. Phone: 49 345 5526353. Fax: 49 345 5527010. E-mail: ute.lechner{at}mikrobiologie.uni-halle.de

{triangledown} Published ahead of print on 5 February 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Fachgebiet Technische Biochemie, Technische Universität, 13353 Berlin, Germany.

Applied and Environmental Microbiology, April 2009, p. 1876-1884, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01042-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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