Quantitative Analysis of the Relative Transcript Levels of Four Chlorophenol Reductive Dehalogenase Genes in Desulfitobacterium hafniense PCP-1 Exposed to Chlorophenols

Culture conditions.D. hafniense PCP-1 (ATCC 700357) was cultivated anaerobically in either 70-ml serum vials or 1-liter bottles containing, respectively, 35 or 500 ml of mineral salt medium with pyruvate and yeast extract as described previously (8). In all experiments, the medium was inoculated with 5% (vol/vol) of an exponentially growing culture. For 2,4,6-TCP and 3,5-DCP exposure experiments, strain PCP-1 was grown without chlorophenol for two successive transfers. The last culture was used to inoculate three cultures (500 ml) without chlorophenol. After an incubation at 30°C for 30 h (optical density at 600 nm [OD₆₀₀], 0.25 to 0.27), the first culture received 2,4,6-TCP at 50 μM (final concentration) and the second 3,5-DCP at 60 μM (final concentration). No chlorophenol was added to the third culture (unexposed culture). Aliquots of the cultures (20 ml) were transferred into 70-ml sterile serum bottles. After various incubation periods (0, 4, 8, 18, 28, and 44 h) at 30°C, a 2-ml sample was collected for measuring the OD₆₀₀ and the chlorophenol concentrations. For the exposure experiments with different concentrations of 2,4,6-TCP or 3,5-DCP and with PCP, strain PCP-1 was grown without chlorophenol for three successive transfers. The last culture (500 ml) was incubated at 30°C for 30 h (OD₆₀₀, 0.25 to 0.27). A volume of 20 ml of the cultures was transferred into 70-ml sterile anaerobic serum bottles. PCP was then added at 30 μM, whereas 2,4,6-TCP was added at 0.125, 5, 25, and 50 μM and 3,5-DCP at 0.15, 3, 6, 30, and 60 μM (final concentration). The cultures were incubated at 30°C for 12 h. For the temperature experiment, the bottles were incubated for 3 h at 22, 30, or 37°C prior to addition of 3,5-DCP at 60 μM. The bottles were further incubated for 12 h at these temperatures. For each type of experiment, an additional bottle was treated similarly but without chlorophenol and served as an unexposed culture.

For analysis of chlorophenols, 0.5 ml of acetonitrile containing 0.33% (vol/vol) acetic acid was added to a 1-ml culture sample. The mixture was centrifuged for 5 min at 5,000 × g, and the supernatant was collected for high-pressure liquid chromatography (HPLC) analysis as described previously (10).

RNA extraction and RT-qPCR.Cells were collected from a 12-ml culture sample by centrifugation at 6,000 × g for 4 min at 4°C and dispersed in 450 μl of ice-cold RNAwiz reagent (Ambion, Austin, TX). Total RNA was extracted using a RiboPure Bacteria kit (Ambion) according to the manufacturer's recommendations. Contaminant DNA was removed by two successive treatments with a DNA-free kit (Ambion). Primers (Table 1) were designed using the D. hafniense DCB-2 genomic sequence currently deposited in the NCBI GenBank database (www.ncbi.nlm.nih.gov; accession number NC_011830) to target the genes cprA2, cprA3, cprA4, and cprA5 and the gene carrying the RNA polymerase beta subunit rpoB (locus tags, Dhaf_0711, Dhaf_0713, Dhaf_0689, Dhaf_0696, and Dhaf_0414, respectively). Each of the primer sets was tested for specificity and efficiency of amplification by qPCR with serial dilutions of strain PCP-1 total DNA as the standard. The PCR efficiency (E) for each primer pair was calculated from the slope of the standard curve according to the following formula: E = (10^−1/slope) − 1. The RT-qPCR analyses were performed using an iScript One-Step RT-PCR kit with SYBR green (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada) according to the manufacturer's instructions, with 50 ng total RNA or diethylpyrocarbonate-treated water (negative control) and 10 nM each primer (forward and reverse) in a 25-μl volume. Thermocycling conditions for reverse transcription and PCR amplification were as follows: 10 min at 50°C and 5 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 55°C. Transcript levels of the cprA genes were calculated by relative quantification using the ΔΔCT method described by Livak and Schmittgen (12), with rpoB as the normalizing gene (4, 13). Cultures exposed to chlorophenols were compared to unexposed cultures. Threshold cycle (C_T) expression data were reported as 2^−(ΔΔCT). An induction factor higher than 10 with respect to the unexposed cultures was arbitrarily set as representing significant induction. Variations in rpoB C_T were minimal (1 C_T or less) whatever the PCP-1 culture conditions.

Exposure to 2,4,6-TCP, 3,5-DCP, and PCP.Dechlorination of 2,4,6-TCP and 3,5-DCP occurred after an 8-h lag period in both cases. 2,4,6-TCP was almost entirely converted into 2,4-DCP after 18 h and into 4-chlorophenol (4-CP) after 44 h (Fig. 1 A). 3,5-DCP was almost entirely converted into 3-CP after 28 h (Fig. 2 A). Relative to the results seen with unexposed cultures, cprA3 transcription was upregulated 400-fold within 4 h in 2,4,6-TCP-amended cultures (Fig. 1B). Upregulation reached its highest (18,000-fold) level within 18 h and then decreased. Similarly, cprA2 transcription was upregulated (40-fold) within 4 h; upregulation reached a maximum (160-fold) within 18 h and then decreased. cprA5 transcription was upregulated 60-fold after 18 h, and upregulation reached 1,200-fold after 44 h (Fig. 1B). Exposure to 3,5-DCP did not significantly affect the transcript levels of cprA2 and cprA3 (Fig. 3 B). cprA5 transcription upregulation reached 100-fold within 4 h and reached its highest level (1,200-fold) within 18 h (Fig. 2B). No significant increase in cprA4 transcript levels was observed after exposure to either 2,4,6-TCP or 3,5-DCP.

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Fig. 1.

Kinetics of dehalogenation of 2,4,6-TCP and relative quantification of transcripts of cprA2, cprA3, cprA4, and cprA5 in 2,4,6-TCP-amended PCP-1 cultures. (A) 2,4,6-TCP (50 μM) was added to exponential cultures at 30°C. The cultures were then sampled at different times in hours (x axis) to measure the concentrations of chlorophenols and to extract total RNA. Diamonds, 2,4,6-TCP; squares, 2,4-DCP; triangles, 4-CP. (B) RT-qPCR were performed for each cprA gene. The relative quantification data represent ratios of 2^−(ΔΔCT) values between the chlorophenol-exposed cultures and the unexposed cultures. Relative values that fall between 0.1 and 10 (the gray zone) were not considered to represent significant differences in the results obtained for the unexposed cultures, taking the standard deviation into account. Results represent data from duplicated separate experiments.

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Fig. 2.

Kinetics of dehalogenation of 3,5-DCP and relative quantification of transcripts of cprA2, cprA3, cprA4, and cprA5 in 3,5-DCP-amended PCP-1 cultures. 3,5-DCP (60 μM) was added to exponential cultures at 30°C. See Fig. 1 legend for details. Diamonds, 3,5-DCP; squares, 3-CP.

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Fig. 3.

Effect of the concentrations of 2,4,6-TCP and 3,5-DCP on cprA3 and cprA5 transcript levels. Different concentrations of 2,4,6-TCP (A) or 3,5-DCP (B) were added to exponentially growing cultures that were incubated at 30°C for 12 h. Total RNA was extracted from cultures, and RT-qPCR analyses were performed. Results represent data from duplicated separate experiments.

Concentrations of naturally occurring chlorophenols in environment are minute (at approximately picomolar levels) (14) but can reach millimolar levels in contaminated soil (11). To see the effect of the chlorophenol concentrations on the transcript levels of cprA3 and cprA5, cultures were exposed for 12 h to 2,4,6-TCP at concentrations ranging from 0.125 to 50 μM and to 3,5-DCP at concentrations ranging from 0.15 to 60 μM. The cprA3 transcript level increased by 1,000-fold at the lowest concentration of 2,4,6-TCP (0.125 μM) compared to unexposed culture results and reached a maximum (4,000-fold increase) at 25 μM (Fig. 3A). The cprA5 transcript level was upregulated more than 200-fold in the presence of 3,5-DCP at 0.15 μM. The induction leveled off at 6 to 60 μM with a 900-fold increase (Fig. 3B). These results suggest that dehalogenating activities in strain PCP-1 would be induced in soil or water environments upon an addition of a small amount of chlorophenols.

Exponentially growing cultures were incubated with PCP for 12 h. Bouchard et al. (2) showed that PCP was completely transformed in 3,4,5-TCP in less than 12 h, followed by a 36-hour lag period before 3,4,5-TCP dehalogenation began to occur. The transcript levels of cprA2 and cprA3 increased by 700-fold and 33,500-fold, respectively. cprA4 transcription was upregulated by about 13-fold, while the cprA5 transcript level was not significantly affected (data not shown).

Effect of temperature.Thibodeau et al. (17) showed that the growth temperature can affect the production of CprA5 in strain PCP-1. Although the optimal growth temperature for strain PCP-1 is 38°C, the CprA5-specific activity was 2.2- and 9.6-fold higher in strain PCP-1 cultured at 22°C than at 30°C and at 37°C, respectively (17). To determine the effect of the temperature on the cprA5 transcript level, exponentially growing cultures were placed at 22, 30, and 37°C and exposed to 3,5-DCP for 12 h. The cprA5 transcript levels were similar at 30°C and 37°C but were 50-fold higher than those seen at 22°C (data not shown). This suggests a posttranscriptional regulation of CprA5 expression (such as transcript stability) at 22°C. As observed at 30°C, no significant upregulation of cprA2, cprA3, and cprA4 transcript levels was observed at 22°C or 37°C after exposure to 3,5-DCP.

cprA2 and cprA3.cprA2 and cprA3 transcript levels were upregulated in 2,4,6-TCP- and PCP-amended cultures but not in 3,5-DCP-amended cultures. In 2,4,6-TCP-amended cultures, the two genes showed similar patterns of expression over time. Upregulation was clearly apparent in both cases 8 h after addition of 2,4,6-TCP. However, 2,4,6-TCP dechlorination was observed only after this period. Similarly, addition of 3-chloro-4-hydroxyphenylacetate (3Cl4OHPA) to D. dehalogenans cultures increased the expression of cprA, a gene encoding the 3Cl4OHPA RDase, by 15-fold after 30 min, but significant dechlorination was observed only after 2 h (16). These results suggest that a lag period could be needed for de novo protein synthesis, which is consistent with a lack of dechlorinating activity in cultures supplemented with chloramphenicol (3, 18). Furthermore, other factors may have to be produced before dechlorination can occur. Such factors could include components of the organohalide respiration chain and proteins involved in the maturation and translocation of the enzyme. CprA proteins contain cobalamin and iron-sulfur cofactors, as well as a twin-arginine signal sequence that may be involved in targeting the enzyme to the TAT secretion pathway for translocation across the cytoplasmic membrane in a folded conformation (9, 15, 19).

Our results showed that more than one chlorophenol can upregulate cprA2 and cprA3. It has previously been shown that, in D. hafniense strain DCB-2, 3Cl4OHPA can induce expression of cprA1, cprA2, and cprA3 and that several haloge- nated phenolic compounds such as 2,4-DCP, 2Br-4-CP, and 3Cl4OHPA can bind to D. hafniense DCB-2 CprK1, a CRP-FNR-type transcriptional regulator involved in transcriptional activation of cprA1 in strain DCB-2 (6, 7). Broad CprK substrate-binding specificity could explain the finding that several chlorophenols (PCP, 2,4,6-TCP, 2,3,4-TCP, 2,3,5-TCP, 2,6-DCP, and 2,4-DCP) can act as inducers of ortho-dechlorinating activity in strain PCP-1 (3).

Based on the fact that cprA2 and cprA3 are upregulated similarly and that their deduced amino acid sequences are highly similar (69%), this suggests that the cprA2 gene product is involved in ortho-dechlorination of highly chlorinated phenols.

cprA5.3,5-DCP upregulated only cprA5 expression in PCP-1 cultures. The kinetics of induction of 3,5-DCP dechlorination was similar to that observed with 2,4,6-TCP. Upregulation (580-fold) of cprA5 expression was clearly apparent at 8 h after addition of 3,5-DCP, but 3,5-DCP dechlorination was observed only after this period. In the 2,4,6-TCP-amended cultures, cprA5 upregulation occurred concomitantly with the appearance of 2,4-DCP, suggesting that this molecule can induce cprA5 expression. The levels of upregulation induced by 2,4-DCP (1,200-fold increase in transcription) were similar to the levels measured in the 3,5-DCP-amended cultures.

cprA4.cprA4 transcription was not upregulated in the presence of 2,4,6-TCP or 3,5-DCP and was barely (13-fold) upregulated above the arbitrary limit threshold in the presence of PCP. Phylogenic analyses showed that deduced amino acid sequences of cprA2, cprA3, and cprA4 grouped together (69 to 76% similarity) (19), suggesting related activities, such as the dehalogenation of highly chlorinated compounds, among these 3 Rdases, although further experiments are needed to confirm this.

In conclusion, the findings with respect to transcriptional upregulation of cprA3 by 2,4,6-TCP and PCP and of cprA5 by 3,5-DCP concur with the CprA3- and CpA5-specific activities toward different chlorophenols, where CprA3 preferentially dechlorinates highly chlorinated phenols and CprA5 preferentially dechlorinates DCPs. D. hafniense strain PCP-1 was isolated from a PCP-degrading microbial consortium (2). The presence of several genes involved in dehalogenation of highly chlorinated phenols and their induction by several chlorophenols would allow strain PCP-1 (and the consortium) to response efficiently to the presence of these toxic compounds by producing versatile enzymes to carry the dehalogenation reactions. The dehalogenation products (DCPs) could in turn induce expression of cprA5 for the dehalogenation of the lesser chlorinated phenol to monochlorophenols.