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.Highly chlorinated aromatic compounds, like polychlorinated benzenes, dibenzo-p-dioxins and -furans, and polychlorinated biphenyls (PCB) persist in the environment for long periods, tend to accumulate in food chains, and are ubiquitously distributed. Many freshwater and marine sediments worldwide have been polluted by high concentrations of these compounds (4, 7, 43). Under reducing conditions often prevailing in sediments, reductive dehalogenation is considered the main biological process for the natural attenuation of highly chlorinated compounds. Several anaerobic bacteria are known to grow by respiratory dehalogenation with chlorinated compounds (for a review, see reference 40). Physiological studies with pure cultures of "Dehalococcoides" spp. and related bacteria of subphylum II of the Chloroflexi have shown that they are highly specialized in using reductive dechlorination as a respiratory process (3,12,13,30,41). The genome sequences of "Dehalococcoides ethenogenes" strain 195 and Dehalococcoides sp. strain CBDB1 confirmed their metabolic restriction to respiratory dehalogenation (24, 38) and revealed the presence of 17 and 32 reductive dehalogenase homologous (rdh) genes, respectively, some...

Role of “Dehalococcoides” spp. in the Anaerobic Transformation of Hexachlorobenzene in European Rivers

“…5A). A similar response was observed during 1,2,3-TCB and 1,2,4-TCB dechlorination by strain CBDB1 (44). Throughout the experiment, cbrA transcript levels were found to be 3-to 50-fold higher than those at the start of the incubation.…”

“…in Flix river sediment possessed rdhA genes corresponding to those previously identified in the genome of strain CBDB1 (44). Analysis of 57 T-RFs in the sample taken in the third week of the experiment showed that the Flix sediment batches contained 19 of the 32 rdh genes from strain CBDB1 (Fig.…”

“…Terminal restriction fragment length polymorphism (T-RFLP), sequencing, and qPCR were used to detect rdh genes in the genome of strain CBDB1 in sediment microcosms. As described previously by Wagner and coworkers, 13 degenerate primer pairs were used to amplify specific fragments of catalytic-subunit-encoding rdh genes (rdhA) from strain CBDB1 (44). T-RFLP analysis of 6-carboxyfluorescein (FAM)-labeled fragments amplified from the sample taken in the third week of HCB dechlorination in Flix sediment microcosms was performed by Dr. Van Haeringen Laboratorium b.v. (Wageningen, The Netherlands).…”

“…cDNA for the detection of rdh gene expression was synthesized with random hexamers from 0.1 g of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The expression of two reductive dehalogenase-encoding genes of strain CBDB1, namely, cbrA (CAI82345.1) and cbdbA1624 (CAI83644.1), was followed in experiments addressing the effect of temperature on HCB transformation via RT-qPCR using an iQ5 iCycler (Bio-Rad, Veenendaal, The Netherlands) with the primers and thermocycling program as previously described (44 (44) showed that cbdbA1624 is expressed at levels similar to those of cbrA by strain CBDB1 in the presence of 1,2,4-TCB. Expression of both cbrA and cbdbA1624 was found to be induced-compared to the other rdh genes of strain CBDB1-during 1,2,3-TCB and/or 1,2,4-TCB dechlorination.…”

“…To further investigate the involvement of specific reductive dehalogenases in the transformation of HCB, we also measured transcript levels of 1,2,3,4-TeCB and 1,2,3-TCB reductase-encoding cbrA and cbdbA1624, the latter of which is potentially involved in 1,2,4-TCB degradation (44). Transcript copy numbers were normalized to 16S rRNA and examined during the transformation of HCB at the two different temperatures (Fig.…”