Evaluation of the inhibitory effects of chloroform on ortho-chlorophenol- and chloroethene-dechlorinating Desulfitobacterium strains
© Futagami et al.; licensee Springer. 2013
Received: 30 March 2013
Accepted: 21 May 2013
Published: 27 May 2013
Organohalide-respiring Desulfitobacterium strains are believed to play an important role in the bioremediation and natural attenuation of chlorinated aliphatic and aromatic hydrocarbons. However, several studies have reported that chloroform significantly inhibits microbial reductive dechlorination of chloroethene. In this study, we examined the effect of chloroform on several Desulfitobacterium strains, including ortho-chlorophenol-dechlorinating Desulfitobacterium dehalogenans JW/IU-1 and Desulfitobacterium hafniense DCB-2, and also the chloroethene-dechlorinating strain D. hafniense TCE1. In medium containing 3-chloro-4-hydroxyphenylacetate as an electron acceptor, chloroform inhibited the growth of strains JW/IU-1 and DCB-2. Although chloroform did not directly inhibit dechlorination of 3-chloro-4-hydroxyphenylacetate by resting cells, cells cultivated with chloroform showed decreased dechlorination activity. Moreover, transcription of the gene encoding the reductive dehalogenase CprA decreased significantly in cells cultivated with chloroform. These results indicate that chloroform inhibits the growth and dechlorination activity of strains JW/IU-1 and DCB-2 via inhibition of cprA transcription. In contrast, cultivation of strain TCE1 in the presence of chloroform gave rise to a PceA reductive dehalogenase gene-deletion variant of strain TCE1; a similar phenomenon was observed in our previous study of chloroethene-dechlorinating D. hafniense strain Y51. Our results suggest that chloroform extensively inhibits the dechlorination activity of Desulfitobacterium strains, and that the inhibitory mechanism appears to differ between ortho-chlorophenol dechlorinators and chloroethene dechlorinators.
KeywordsDesulfitobacterium Reductive dechlorination Organohalide respiration Growth inhibition Chloroform Chlorophenol Chloroethene
Organohalide respiration is an anaerobic process in which a halogenated organic compound serves as the electron acceptor. Various organohalide-respiring bacteria (OHRB) have been applied to the bioremediation of toxic chlorinated hydrocarbons in anaerobic environments (Smidt and de Vos 2004;Löffler and Edwards 2006;Maphosa et al. 2010). In this process, organohalides are reductively dehalogenated. For example, tetrachloroethene (PCE) is successively converted to trichloroethene (TCE), dichloroethene (cis-dichloroethene or trans-dichloroethene), vinyl chloride (VC), and finally nontoxic ethene.
Species of the genus Desulfitobacterium are some of the most frequently identified OHRB, and can utilize a variety of electron acceptors, such as nitrate, sulfite, fumarate, humic acids, and organohalides (Villemur et al. 2006). Most Desulfitobacterium isolates can respire with ortho-chlorophenol and/or chloroethene. For example, Desulfitobacterium hafniense strain DCB-2 and Desulfitobacterium dehalogenans strain JW/IU-1 respire with ortho-chlorophenols such as 3-chloro-4-hydroxyphenylacete (3-Cl-4-OHPA), whereas D. hafniense strains TCE1 and Y51 respire with PCE and TCE (Madsen and Licht 1992;Utkin et al. 1994;Gerritse et al. 1999;Suyama et al. 2001).
The dehalogenation reaction is catalyzed by reductive dehalogenase, which is the terminal reductase of the organohalide respiratory chain. The dehalogenation spectrum of each OHRB is believed to be determined by the type of this key enzyme. Both D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2 possess ortho-chlorophenol reductive dehalogenase (CprA), for which the corresponding gene cluster (consisting of cprA, cprB, cprC, cprD, cprE, cprK, cprT, and cprZ) has been identified (Smidt et al. 2000;Christiansen et al. 1998;Kim et al. 2012). On the other hand, D. hafniense strains TCE1 and Y51 possess the PCE reductive dehalogenase (PceA) gene cluster, which consists of pceA, pceB, pceC, and pceT (Maillard et al. 2005;Suyama et al. 2002;Furukawa et al. 2005). The pce genes are surrounded by two copies of nearly identical insertion sequence (IS) elements that contain a gene homologous to the IS256 family transposase; therefore, the pce genes can function as a typical composite transposon. In fact, several types of circular DNAs resulting from homologous recombination between two homologous IS elements and transposase-mediated excision and circularization have been identified in strains TCE1 and Y51 (Maillard et al. 2005;Futagami et al. 2006a;Duret et al. 2012).
The OHRB play a significant role in the detoxification of chlorinated hydrocarbons. However, chloroform (CF, trichloromethane), which is a common environmental pollutant that has both biotic and abiotic origins, reportedly inhibits microbial reductive dechlorination of chloroethene (Cappelletti et al. 2012). Bagley et al. (2000) showed that CF at a concentration of 4 μM completely inhibits degradation of PCE by a microcosm, while Duhamel et al. (2002) showed that a chloroethene-dechlorinating microcosm containing Dehalococcoides species is inhibited by 2.5 μM CF, resulting in accumulation of VC. Inhibitory effects of CF on the chloroethene dechlorination activity of the OHRB isolate Dehalococcoides mccartyi strain 195, and on the activity of purified PceA reductive dehalogenase from Sulfurospirillum multivorans have also been reported (Maymó-Gatell et al. 2001;Neumann et al. 1996;Löffler et al. 2013). In the case of D. hafniense strain Y51, we found that two different PCE-nondechlorinating variants become dominant in the presence of CF. One of these variants (designated the LD variant) lost all pce genes due to homologous recombination between the two IS elements. The other nondechlorinating variant lost one IS element located upstream of the pce gene cluster, in which a portion of the promoter region is located. This type of variant, designated as SD variant, cannot express pce genes. Both of these variants, but especially the LD variant, become dominant in the presence of CF, which inhibits the growth of wild type strain Y51 at concentrations as low as 1 μM, while both the LD and SD variants can grow normally at CF concentrations as high as 1 mM (Futagami et al. 2006b).
Although the effects of CF on the reductive dechlorination of chloroethene have been studied, how CF affects ortho-chlorophenol dechlorination remains to be elucidated. We therefore investigated the effect of CF on the growth, dechlorination activity, the genetic stability and transcription of the cprA gene in the ortho-chlorophenol-dechlorinating bacteria D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2. In addition, we investigated the effect of CF on the chloroethene-dechlorinating bacterium D. hafniense strain TCE1, and compared the results to those from our previous study on D. hafniense strain Y51. We report here that CF strongly inhibits the dechlorination activity of Desulfitobacterium strains, and discuss the sigificance of the inhibitory effect of CF on ortho-chlorophenol-dechlorinating and chloroethene-dechlorinating Desulfitobacterium strains.
Materials and methods
Strains and cultivation
Desulfitobacterium dehalogenans strain JW/IU-1 (DSM 9161) and D. hafniense strains DCB-2 (DSM 10664) and TCE1 (DSM 12704) were obtained from the DSMZ microbe collection. Desulfitobacterium hafniense strain Y51 was previously isolated in our laboratory (Suyama et al. 2001). Strains JW/IU-1, DCB-2, TCE1, and Y51 were grown anaerobically at 30°C in MMYP medium (45.9 mM K2HPO4, 8.8 mM KH2PO4, 1.7 mM sodium citrate, 0.4 mM MgSO4 · 7H2O, yeast extract [2.0 g/L], 68.2 mM sodium pyruvate, and 4.0 μM resazurin sodium salt, pH 7.2) with 10 mM 3-Cl-4-OHPA or 5 mM sodium fumarate as an electron acceptor. CF was diluted in N,N-dimethylformamide and added to the culture medium to a final concentration of 1, 10, or 100 μM. Growth of all strains was assessed by optical density (OD) at 660 nm. Growth was assessed at least two times independently.
Dechlorination of 3-Cl-4-OHPA by resting cells
Resting cells were prepared from cultures of strains JW/IU-1 and DCB2 grown in MMYP medium with 10 mM 3-Cl-4-OHPA and with or without 100 μM CF. The strains JW/IU-1 and DCB-2 were grown to late-log phase (OD660 = 0.2 and 0.4, respectively) and the cells were harvested by centrifugation at 4,000 × g for 10 min at 4°C. The cell pellet was washed with buffer (45.9 mM K2HPO4 and 8.8 mM KH2PO4, pH 7.5) and recentrifuged twice under the same conditions. The cells were then resuspended in phosphate buffer to an OD660 of 0.3. Dechlorination of 3-Cl-4-OHPA was measured in a 25-ml glass vial containing 5.0 ml of resting cell solution (OD660 = 0.25) containing 20 mM sodium pyruvate and 1.0 mM 3-Cl-4-OHPA. Each vial was sealed with a butyl rubber stopper and crimped. CF was added to a final concentration of 100 μM by syringe. All manipulations were performed under anaerobic conditions using centrifuge tubes with sealing caps (Nalgene, Rochester, NY) and an anaerobic grove box with an atmosphere of 85% N2, 5.0% CO2, and 10% H2. The reaction mixtures were incubated at 30°C with shaking at 120 rpm. The reaction solutions were collected with a syringe and filtered through a 0.2-μm PTFE filter (Millex-LG, Millipore, Bedford, MA). The 3-Cl-4-OHPA remaining in solution was quantified by high-performance liquid chromatography (HPLC) on a system equipped with a Waters 2487 UV detector operated at 280 nm (Waters, Milford, MA) and a YMC Pack Pro C18 RS column (YMC, Kyoto, Japan). The mobile phase was acetonitrile-water-acetic acid (30:70:1), and the column was eluted isocratically at a flow rate of 1 ml/min. Dechlorination activity was measured in triplicate samples and the results shown are the mean and standard deviation.
Stability of the reductive dehalogenase genes
Sequences of primers used in this study
Strains TCE1 and Y51 were cultivated in MMYP medium with 5 mM fumarate and with or without 100 μM CF and then harvested at stationary phase. Isolation of genomic DNA and Southern blot analysis were performed as described above. The DIG-labeled DNA probe used to detect the pceA gene was amplified as described previously (Futagami et al. 2006b).
Northern blot analysis of cprA transcripts
Strains JW/IU-1 and DCB-2 were cultivated in MMYP medium with 10 mM 3-Cl-4-OHPA and with or without 100 μM CF and then harvested at the logarithmic phase. Total RNA was isolated from cells using RNAiso reagent (Takara, Shiga, Japan) according to the manufacturer’s protocol. The concentration of RNA was determined by measuring the absorbance at 260 nm with a spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan). Total RNA (30 μg) was electrophoresed on a 1.0% agarose gel with 18% (vol/vol) formaldehyde and then transferred onto a Hybond N membrane (Amersham Biosciences, Buckinghamshire, UK). Hybridization with DIG-labeled cprA DNA probes and subsequent detection with NBT/BCIP were carried out according to the manufacturer’s instructions. The cprA gene probe for strain JW/IU-1 and the cprA gene probe for strain DCB-2 were amplified using the primer sets DdehalogF2-DdehalogR2 and DCB2F1-DCB2-R1, respectively (Table 1).
Effect of CF on the growth of D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2
Effect of CF on dechlorination of 3-Cl-4-OHPA by D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2
Effect of CF on the stability and transcription of cprA in D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2
In contrast, CF did affect the transcription of the cprA gene in strains JW/IU-1 and DCB-2 (Figure 3). The RNAs extracted from strains JW/IU-1 and DCB-2 cultivated in medium containing 3-Cl-4-OHPA and with or without 100 μM CF were subjected to northern blot analysis using cprA as a probe. The signal intensity of the cprA transcript from cells of strains JW/IU-1 and DCB-2 grown in the presence of CF was significantly lower than that of cells grown in the absence of CF. This result agreed with the reduction in 3-Cl-4-OHPA dechlorination activity of strains JW/IU-1 and DCB-2 when cells were grown in the presence of 100 μM CF (Figure 2).
Effect of CF on the growth and pceA-stability of D. hafniense TCE1
Next, we examined the effect of CF on the stability of the pce gene cluster in D. hafniense TCE1, and compared the results to those obtained previously with D. hafniense Y51 (Figure 4B). Genomic DNA was extracted from cells cultivated with or without 100 μM CF and then subjected to Southern blot analysis using pceA as a probe. Southern blot analysis was used previously to detect the varieties of genetic alterations in pce gene clusters caused by IS elements in strains TCE1 and Y51 (Maillard et al., 2005;Futagami et al. 2006a;Futagami et al. 2006b) (Figure 4C). In the case of wild type strain Y51, a 5.0 kb fragment was detected in cells cultivated in the absence of CF, but in cells cultivated with CF the intensity of this 5.0 kb band was significantly reduced and a 3.5 kb band was detected (Figure 4B). The 3.5 kb fragment was derived from a PCE-nondechlorinating SD variant that emerged following deletion of the IS element located upstream of the pceA gene, as described previously (Figure 4C) (Futagami et al. 2006b).
In the case of strain TCE1, DNA bands of 9.4 kb and 6.0 kb were detected when the cells were cultivated in the absence of CF. The 9.4-kb DNA fragment was derived from chromosomal DNA containing pceA, whereas the 6.0-kb DNA fragment was derived from circular DNA containing pceA, which was generated by homologous recombination between the two identical IS elements, as shown in Figure 4C (Maillard et al. 2005). The intensity of these two pce-containing bands was significantly reduced when strain TCE1 was cultivated in the presence of CF, indicating that the pce-deletion mutant became dominant after the cells were cultivated in the presence of CF. Thus, the pce gene cluster is very unstable, and is deleted in the presence of CF in both strains Y51 and TCE1.
In this study, we investigated the effects of CF on the ortho-chlorophenol-dechlorinating bacteria D. dehalogenans strain JW/IU-1 and D. hafniense strain DCB-2. The growth of both strains was inhibited by CF at a concentration of 100 μM in medium containing 3-Cl-4-OHPA as the electron acceptor (Figure 1). It is noteworthy that 100 μM CF did not directly inhibit the dechlorination of 3-Cl-4-OHPA by resting cells (Figure 2). However, the dechlorination activity of cells cultivated in the presence of 100 μM CF was lower than that of cells cultivated in the absence of CF. This reduction in dechlorination activity was due to decreased transcription of cprA in the presence of CF (Figure 3). These results suggest that inhibition of growth mediated by CF in medium containing 3-Cl-4-OHPA is due to inhibition of CprA expression, as this protein is essential for organohalide respiration utilizing 3-Cl-4-OHPA.
In strains JW/IU-1 and DCB-2, transcription of the cpr genes is strictly regulated by CprK, which is a member of the cAMP-binding protein-fumarate nitrate reduction regulatory protein (CPR-FNR) family of transcriptional factors (Pop et al. 2004;Gabor et al. 2006). The interaction of a chlorinated aromatic substrate such as 3-Cl-4-OHPA with the effector domain of CprK triggers binding of CprK to a DNA sequence called the dehalobox. Binding of CprK to the dehalobox leads to transcriptional activation of the cpr gene cluster. Thus, CF might act as a competitive inhibitor of 3-Cl-4-OHPA, keeping CprK in an inactivated state.
The above-described mechanism through which CF inhibits the growth of Desulfitobacterium is supported by the observed differences in the degree that CF inhibited the growth of strain JW/IU-1 under different cultivation conditions. In the presence of 100 μM CF, growth rate of JW/IU-1 was inhibited in medium containing 3-Cl-4-OHPA, but not in medium containing fumarate as the electron acceptor, which may be explained by that the expression of CprA is not required for fumarate respiration. In fact, it was demonstrated that the transcriptional level of cprA in the medium containing fumarate as an electron acceptor was significantly lower than that in a medium containing 3-Cl-4-OHPA as an electron acceptor (Smidt et al. 2000). On the other hand, CF inhibited the growth of strain DCB-2 to a greater degree than strain JW/IU-1. Even at a concentration as low as 1 μM, CF inhibited the growth of strain DCB-2 in medium containing either 3-Cl-4-OHPA or fumarate. Moreover, CF inhibited the growth rate of strain JW/IU-1, whereas CF caused both the extended lag phase and the reduced cell yield of strain DCB-2. This result indicates that a direct toxic effect of CF on the overall fitness of the cells of strain DCB-2 also present. Similar inhibitory effects of organic solvents have been observed for anaerobic bacteria (Duldhardt et al. 2007;Duldhardt et al. 2010).
CF inhibited the growth of the PCE- and TCE-dechlorinator D. hafniense strain TCE1. This result was similar to that observed in our previous study involving D. hafniense strain Y51 (Futagami et al. 2006b). Southern blot analysis using pceA as a probe showed that the signal intensity associated with pceA decreased significantly when cells of strain TCE1 were cultivated in the presence of CF, indicating that the pceA-deletion variant became dominant. Although the pce genes of strains TCE1 and Y51 are very similar (>99%, including the intergenic regions) (Maillard et al. 2005;Futagami et al. 2006a) and CF had a similar inhibitory effect on the growth of both strains, one difference was the occurrence of the circular Tn-Dha1 DNA element containing the entire pce gene cluster (Figure 4B and C). The band derived from the Tn-Dha1 element formed by homologous recombination between the two identical IS elements was detected by Southern blot analysis, as described previously (Maillard et al. 2005). Strain Y51 also has the same type of circular element, designated TnDesp1 (Futagami et al. 2006a), with a predicted band size on Southern blotting of ca. 6.5 kb. However, no 6.5-kb DNA band was detected under the cultivation conditions used in this study, indicating that the efficiency of excision by homologous recombination between the IS elements and/or the stability of the circular elements of strains TCE1 and Y51 might be different.
In conclusion, CF had a negative effect on all the Desulfitobacterium strains tested, including D. dehalogenans strain JW/IU-1, D. hafniense strain DCB-2, and D. hafniense strain TCE1. Because the Desulfitobacterium play a significant role in microbial reductive dechlorination, the presence of CF in the environment could be detrimental to bioremediation efforts aimed at other halogenated hydrocarbons. The degradation of CF through organohalide respiration and fermentation by Dehalobacter spp. was recently reported (Grostern et al. 2010;Lee et al. 2012). The results of these studies could be important for developing effective strategies for the bioremediation of halogenated compounds in anaerobic environments.
This work was supported in part by a Ministry of Education, Science, Sports and Culture Grant-in-Aid for Scientific Research (C) (no. 22580382, to K.F.). The cost of publication was supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University.
- Bagley DM, Lalonde M, Kaseros V, Stasiuk KE, Sleep BE: Acclimation of anaerobic systems to biodegrade tetrachloroethene in the presence of carbon tetrachloride and chloroform. Water Res 2000, 34: 171–178. 10.1016/S0043-1354(99)00121-9View ArticleGoogle Scholar
- Cappelletti M, Frascari D, Zannoni D, Fedi S: Microbial degradation of chloroform. Appl Microbiol Biotechnol 2012, 96: 1395–1409. 10.1007/s00253-012-4494-1PubMedView ArticleGoogle Scholar
- Christiansen N, Ahring BK, Wohlfarth G, Diekert G: Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense . FEBS Lett 1998, 436: 159–162. 10.1016/S0014-5793(98)01114-4PubMedView ArticleGoogle Scholar
- Duhamel M, Wehr SD, Yu L, Rizvi H, Seepersad D, Dworatzek S, Cox EE, Edwards EA: Comparison of anaerobic dechlorinating enrichment cultures maintained on tetrachloroethene, trichloroethene, cis -dichloroethene and vinyl chloride. Water Res 2002, 36: 4193–4202. 10.1016/S0043-1354(02)00151-3PubMedView ArticleGoogle Scholar
- Duldhardt I, Nijenhuis I, Schauer F, Heipieper HJ: Anaerobically grown Thauera aromatica , Desulfococcus multivorans , Geobacter sulfurreducens are more sensitive towards organic solvents than aerobic bacteria. Appl Microbiol Biotechnol 2007, 77: 705–711. 10.1007/s00253-007-1179-2PubMedView ArticleGoogle Scholar
- Duldhardt I, Gaebel J, Chrzanowski L, Nijenhuis I, Härtig C, Schauer F, Heipieper HJ: Adaptation of anaerobically grown Thauera aromatica , Geobacter sulfurreducens and Desulfococcus multivorans to organic solvents on the level of membrane fatty acid composition. Microb Biotechnol 2010, 3: 201–209. 10.1111/j.1751-7915.2009.00124.xPubMed CentralPubMedView ArticleGoogle Scholar
- Duret A, Holliger C, Maillard J: The physiological opportunism of Desulfitobacterium hafniense strain TCE1 towards organohalide respiration with tetrachloroethene. Appl Environ Microbiol 2012, 78: 6121–6127. 10.1128/AEM.01221-12PubMed CentralPubMedView ArticleGoogle Scholar
- Furukawa K, Suyama A, Tsuboi Y, Futagami T, Goto M: Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review. J Ind Microbiol Biotechnol 2005, 32: 534–541. 10.1007/s10295-005-0252-zPubMedView ArticleGoogle Scholar
- Futagami T, Tsuboi Y, Suyama A, Goto M, Furukawa K: Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51. Appl Microbiol Biotechnol 2006, 70: 720–728. 10.1007/s00253-005-0112-9PubMedView ArticleGoogle Scholar
- Futagami T, Yamaguchi T, Nakayama S, Goto M, Furukawa K: Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51. Appl Environ Microbiol 2006, 72: 5998–6003. 10.1128/AEM.00979-06PubMed CentralPubMedView ArticleGoogle Scholar
- Gabor K, Verissimo CS, Cyran BC, Ter Horst P, Meijer NP, Smidt H, de Vos WM, van der Oost J: Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense . J Bacteriol 2006, 188: 2604–2613. 10.1128/JB.188.7.2604-2613.2006PubMed CentralPubMedView ArticleGoogle Scholar
- Gerritse J, Drzyzga O, Kloetstra G, Keijmel M, Wiersum LP, Hutson R, Collins MD, Gottschal JC: Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1. Appl Environ Microbiol 1999, 65: 5212–5221.PubMed CentralPubMedGoogle Scholar
- Grostern A, Duhamel M, Dworatzek S, Edwards EA: Chloroform respiration to dichloromethane by a Dehalobacter population. Environ Microbiol 2010, 12: 1053–1060. 10.1111/j.1462-2920.2009.02150.xPubMedView ArticleGoogle Scholar
- Kim SH, Harzman C, Davis JK, Hutcheson R, Broderick JB, Marsh TL, Tiedje JM: Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction. BMC Microbiol 2012, 12: 21. 2180–12–21 10.1186/1471-2180-12-21PubMed CentralPubMedView ArticleGoogle Scholar
- Lee M, Low A, Zemb O, Koenig J, Michaelsen A, Manefield M: Complete chloroform dechlorination by organochlorine respiration and fermentation. Environ Microbiol 2012, 14: 883–894. 10.1111/j.1462-2920.2011.02656.xPubMedView ArticleGoogle Scholar
- Löffler FE, Edwards EA: Harnessing microbial activities for environmental cleanup. Curr Opin Biotechnol 2006, 17: 274–284. 10.1016/j.copbio.2006.05.001PubMedView ArticleGoogle Scholar
- Löffler FE, Yan J, Ritalahti KM, Adrian L, Edwards EA, Konstantinidis KT, Muller JA, Fullerton H, Zinder SH, Spormann AM: Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi . Int J Syst Evol Microbiol 2013, 63: 625–635. 10.1099/ijs.0.034926-0PubMedView ArticleGoogle Scholar
- Madsen T, Licht D: Isolation and characterization of an anaerobic chlorophenol-transforming bacterium. Appl Environ Microbiol 1992, 58: 2874–2878.PubMed CentralPubMedGoogle Scholar
- Maillard J, Regeard C, Holliger C: Isolation and characterization of Tn -Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1. Environ Microbiol 2005, 7: 107–117. 10.1111/j.1462-2920.2004.00671.xPubMedView ArticleGoogle Scholar
- Maphosa F, de Vos WM, Smidt H: Exploiting the ecogenomics toolbox for environmental diagnostics of organohalide-respiring bacteria. Trends Biotechnol 2010, 28: 308–316. 10.1016/j.tibtech.2010.03.005PubMedView ArticleGoogle Scholar
- Maymó-Gatell X, Nijenhuis I, Zinder SH: Reductive dechlorination of cis -1,2-dichloroethene and vinyl chloride by " Dehalococcoides ethenogenes ". Environ Sci Technol 2001, 35: 516–521. 10.1021/es001285iPubMedView ArticleGoogle Scholar
- Neumann A, Wohlfarth G, Diekert G: Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans . J Biol Chem 1996, 271: 16516–16519.Google Scholar
- Pop SM, Kolarik RJ, Ragsdale SW: Regulation of anaerobic dehalorespiration by the transcriptional activator CprK. J Biol Chem 2004, 279: 49910–49918. 10.1074/jbc.M409435200PubMedView ArticleGoogle Scholar
- Smidt H, de Vos WM: Anaerobic microbial dehalogenation. Annu Rev Microbiol 2004, 58: 43–73. 10.1146/annurev.micro.58.030603.123600PubMedView ArticleGoogle Scholar
- Smidt H, van Leest M, van der Oost J, de Vos WM: Transcriptional regulation of the cpr gene cluster in ortho -chlorophenol-respiring Desulfitobacterium dehalogenans . J Bacteriol 2000, 182: 5683–5691. 10.1128/JB.182.20.5683-5691.2000PubMed CentralPubMedView ArticleGoogle Scholar
- Suyama A, Iwakiri R, Kai K, Tokunaga T, Sera N, Furukawa K: Isolation and characterization of Desulfitobacterium sp. strain Y51 capable of efficient dehalogenation of tetrachloroethene and polychloroethanes. Biosci Biotechnol Biochem 2001, 65: 1474–1481. 10.1271/bbb.65.1474PubMedView ArticleGoogle Scholar
- Suyama A, Yamashita M, Yoshino S, Furukawa K: Molecular characterization of the PceA reductive dehalogenase of Desulfitobacterium sp. strain Y51. J Bacteriol 2002, 184: 3419–3425. 10.1128/JB.184.13.3419-3425.2002PubMed CentralPubMedView ArticleGoogle Scholar
- Utkin I, Woese C, Wiegel J: Isolation and characterization of Desulfitobacterium dehalogenans gen. nov., sp. nov., an anaerobic bacterium which reductively dechlorinates chlorophenolic compounds. Int J Syst Bacteriol 1994, 44: 612–619. 10.1099/00207713-44-4-612PubMedView ArticleGoogle Scholar
- Villemur R, Lanthier M, Beaudet R, Lepine F: The Desulfitobacterium genus. FEMS Microbiol Rev 2006, 30: 706–733. 10.1111/j.1574-6976.2006.00029.xPubMedView ArticleGoogle Scholar
- Wilson K: Preparation of genomic DNA from bacteria. In Current protocols in molecular biology. Edited by: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. New York: Greene Publishing Associates and Wiley Interscience; 1987:241–245.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.