Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.
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.
Wagner
Anke
A
Institut für Biologie/Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
Adrian
Lorenz
L
Kleinsteuber
Sabine
S
Andreesen
Jan R
JR
Lechner
Ute
U
D001426:Bacterial Proteins / Q000096:biosynthesis; D002722:Chlorobenzenes / Q000378:metabolism; D041862:Chloroflexi / Q000201:enzymology* / Q000378:metabolism; D020869:Gene Expression Profiling; D006867:Hydrolases / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013997:Time Factors; D015854:Up-Regulation
D001426:Bacterial Proteins; D002722:Chlorobenzenes; C009947:1,2,4-trichlorobenzene; C036720:trichlorobenzene; D006867:Hydrolases