Growth of Dehalococcoides mccartyi strain CBDB1 by reductive

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Growth of Dehalococcoides mccartyi strain CBDB1 by reductive dehalogenation of brominated benzenes to benzene Anke Wagner,†,‡,∥ Myriel Cooper,‡,∥ Sara Ferdi,‡ Jana Seifert,§ and Lorenz Adrian‡,* †

Applied Biochemistry, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany Department Isotope Biogeochemistry, Helmholtz Centre for Environmental Research − UFZ, Permoserstraße 15, 04318 Leipzig, Germany § Department Proteomics, Helmholtz Centre for Environmental Research − UFZ, Permoserstraße 15, 04318 Leipzig, Germany ‡

ABSTRACT: Brominated aromatics are used in many different applications but occur also naturally. Here, we demonstrate organohalide respiration and growth of Dehalococcoides mccartyi strain CBDB1 with 1,2,4-tribromobenzene, all three dibrominated benzene congeners and monobromobenzene. All bromobenzenes were fully dehalogenated to benzene. Growth yields were between 1.8 × 1014 and 2.8 × 1014 cells per mol of bromide released. Furthermore, a newly designed high-throughput methyl viologen-based photometric microtiter plate assay was established to determine the activity of the reductive dehalogenases in resting cell assays of strain CBDB1 with brominated aromatics as electron acceptors. Activities of 2.8−13.2 nkat per mg total cell protein (0.16−0.8 units per mg total cell protein) were calculated after cultivation of strain CBDB1 on 1,2,4-tribromobenzene. Mass spectrometric analyses and activity assays with whole cell extracts of strain CBDB1 gave strong evidence that four to six reductive dehalogenases were involved in the dehalogenation of all tested brominated benzenes, including the reductive dehalogenases CbdbA80 and CbrA.

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by Flavobacterium sp. strain 39723.15 Tetrabromobisphenol A was completely mineralized in a two-step anaerobic/aerobic process using activated sludge.16 Further examples include several types of naturally occurring bromophenols, which were completely dehalogenated by Desulfovibrio strain TBP-117 and sponge associated anaerobic microorganisms.18 Dehalococcoides strains are capable of reductive dehalogenation of persistent and toxic compounds including chlorinated dioxins, polychlorinated biphenyls, chlorinated ethenes and polychlorinated phenols,19−23 and are commonly used in natural and engineered bioremediation efforts.24−26 However, none of the so far described dehalogenation reactions of halogenated aromatics catalyzed by pure Dehalococcoides cultures have led to nonhalogenated end-products. Recently, Lee et al. described the dehalogenation of tetra- and pentabrominated diphenylethers to the nonbrominated diphenylether in a coculture of Desulfovibrio and Dehalococcoides,27 and found evidence that growth of the Dehalococcoides strain was coupled to polybrominated diphenylether dehalogenation. However, participation of the Desulfovibrio species in the debromination reaction could not be excluded and indeed

INTRODUCTION Brominated organic compounds have a broad spectrum of applications. The polybrominated biphenyls, polybrominated diphenylethers, tetrabromobisphenol A and hexabromobenzene are widely used as flame retardants.1 Other brominated molecules such as 1,4-dibromobenzene (1,4-DBB) and bromoxynil serve as fumigants, intermediates in the synthesis of dyes, as agrochemicals, pharmaceuticals, or herbicides.2,3 Moreover, apart from anthropogenic sources, a wide variety of brominated aromatics are naturally produced, notably in the marine environment by algae,4 hemichordates,5 sponges,6 and molluscs.7 Due to their multiple origins and applications, brominated compounds are widespread contaminants in sediments and water, and may accumulate in the tissues and milk of animals.8−11 Many brominated compounds are considered to have adverse health effects on humans.2,3,12 Several studies described the transformation of brominated compounds by dehalogenation to less brominated products, but only a few studies have described the complete removal of all halogens from brominated compounds. Biotic transformation by microorganisms and fish,13 and abiotic transformations by photolysis leading to less brominated daughter products,14 are known to occur. The pesticide bromoxynil was reductively dehalogenated to 4-hydroxybenzonitrile under anaerobic conditions by Desulfitobacterium chlororespirans.3 Under aerobic conditions bromoxynil was degraded to cyanide and bromide © 2012 American Chemical Society

Received: Revised: Accepted: Published: 8960

January 27, 2012 July 1, 2012 July 16, 2012 July 16, 2012 dx.doi.org/10.1021/es3003519 | Environ. Sci. Technol. 2012, 46, 8960−8968

Environmental Science & Technology

Article

6 °C min−1 to 225 °C, with a final hold for 2 min. Injector and detector temperatures were 250 and 300 °C, respectively. Benzene, which interfered with the solvent peak in the above mentioned method, was analyzed from the headspace of the culture. Two hundred μL of the headspace were removed at each time point of analysis and transferred directly into the injector of a HP5890II GC equipped with a flame ionization detector and a DB-5 column (30 m, 0.32 mm i.d., 0.25 μm film thickness, J&W Scientific, Köln, Germany). Nitrogen (99.999% purity) served as carrier and makeup gas. Injection was done at a column temperature of 35 °C. After 5 min, the column temperature was increased to 60 °C at a rate of 5 °C min−1, then with 30 °C min−1 to 300 °C and a final hold of 10 min was included. Injector and detector temperatures were 240 and 320 °C, respectively. Chloro- and bromobenzene congeners were identified and quantified by injecting single compounds solved in hexane as standards with concentration between 5 and 100 μM. A standard curve for benzene was prepared by spiking capped cultivation bottles filled with 60 mL anoxic water with benzene concentrations of 5−100 μM. Detection limits were at 1−5 μM for all halogenated benzenes. Resting Cell Activity Assay for Reductive Dehalogenases. Dehalogenase activity was analyzed based on an assay described previously,30 using whole nondisrupted cells as catalyst and methyl viologen as an artificial electron donor. The assay solution contained final concentrations of 2 mM titanium(III) citrate, 1.25 mM methyl viologen, 125 mM potassium acetate buffer (pH 5.8), and 625 μM of a brominated or chlorinated benzene. Halogenated benzenes were added from 50 mM stock solutions in acetone. For every tested compound a 10 mL reaction mix was set up in a glass tube. Per 10 mL reaction mix, 125 μL of the 50 mM stock solution in acetone were added. Cells were concentrated 2-fold using a rotary evaporator before adding to the activity test. For that, cultures were filled into evaporator flasks within an anaerobic chamber (Coy Laboratories, Grass Lake, MI) containing nitrogen of 99.999% v/v purity and 2−4% hydrogen. The flasks were connected with the evaporator directly after taking them out of the anaerobic chamber. The evaporator removed oxygen contaminations in the gas phase quickly. After concentration, the flasks were immediately sealed and brought back to the anaerobic chamber. The procedure was monitored by watching the redox indicator resazurin, which was present in all cultures. A change in color was not observed. The protein concentration for the calculation of specific activities was determined using the NanoOrange protein quantitation Kit (Molecular Probes, Leiden, Netherlands) as described previously.31 Two different assay formats were used to measure dehalogenating activity. In the first assay format (“GC-format”), activity was calculated from the concentrations of dehalogenation products analyzed by gas chromatography.30 In the second assay format (“microtiter plate format”), the activity was measured photometrically by following the oxidation of methyl viologen at 578 nm (extinction coefficient: 9.78 mM−1 cm−1) using a microtiter plate reader (Synergy HT, BioTek, Bad Friedrichshall, Germany). For GC-format activity assays, 800 μL of the assay solution was filled into a 2 mL autosampler vial in an anaerobic chamber and the reaction was started by adding 80 μL of the cell suspension. Vials were sealed with Teflon-lined rubber septa and incubated at 30 °C. At time zero or after 4 h of incubation,

Desulfovibrio species have also been described to dehalogenate different bromophenols, and iodophenols.17,28 For a pure Dehalococcoides strain, however, there is no proof of growth using brominated aromatics as respiratory electron acceptors. Here, we investigated the potential of the pure D. mccartyi strain CBDB1 to grow by organohalide respiration using brominated benzenes as respiratory electron acceptors. Bromobenzenes were chosen for this study because we considered bromobenzenes as a simple model electron acceptor for more complex brominated aromatics. CBDB1 was enriched and isolated on chlorobenzenes,19 but later was shown to also dehalogenate chlorinated phenols,23 biphenyls,21 and dioxins.20 Now we show that it also grows on brominated benzenes, and thus may also dehalogenate more complex brominated substrates. Additionally, we investigated the expression of reductive dehalogenases homologous (Rdh) proteins by i) a newly designed photometric 96-well microtiter plate format test, in which activities of Rdh proteins in resting cell suspensions with different halogenated benzenes were measured after incubation with brominated vs chlorinated benzenes, and ii) shotgun proteomics of cultures growing with different halogenated electron acceptors.

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MATERIAL AND METHODS Chemicals. Chlorinated and brominated benzenes were purchased from Sigma-Aldrich (Seelze, Germany) at a purity of 99%. Titanium(III) citrate was prepared as previously described.23 Cultivation. Strain CBDB1 was grown in titanium(III) citrate-reduced, carbonate-buffered synthetic medium with hydrogen as electron donor and 5 mM acetate as carbon source, as previously described.19 After inoculation, the headspace was pressurized with 20% CO2/80% N2 (1.5 bar), and 0.3 bar hydrogen was added to obtain a final pressure of 1.8 bar. Cultivation was performed in the dark at 30 °C without shaking. The electron acceptors 1,2,4-tribromobenzene (TBB), 1,2-dibromobenzene (DBB), 1,3-DBB and 1,4-DBB or monobromobenzene (MBB) were added from 1 M stock solutions in acetone to a final concentration of 50 μM. 1,2,4TBB-dehalogenating cultures were initially set up from cultures grown on 30 μM 1,2,3-trichlorobenzene (TCB). DBB- and MBB-dehalogenating cultures were inoculated initially from cultures grown in the fourth transfer on 1,2,4-TBB. An inoculum percentage of 10% was used leading to starting cell densities between 5 × 105 and 5 × 106 cell mL−1. Cell numbers were quantified after staining with SYBR-green by direct cell counting on agarose-coated slides as previously described.23,29 All cultivations were done in triplicate. Additionally, chemical controls without inoculum, negative growth controls without electron acceptor and positive controls with 30 μM 1,2,3-TCB were setup. Results presented are from cultures of the fifth transfer with 1,2,4-TBB and from the second transfer with dibrominated benzenes or MBB. Analytical Techniques. 1,2,3-TCB and all bromobenzene congeners were extracted from 0.5 mL samples with 1 mL of hexane and analyzed using a Shimadzu GC 14A equipped with a flame ionization detector and a Permabond-FFAP capillary column (25 m, 0.25 mm i.d., 0.25 μm film thickness, Macherey & Nagel, Düren, Germany). Nitrogen (99.999% purity) served as carrier and makeup gas. The following temperature program was used: initial hold at 55 °C for 1 min; increase at a rate of 8961

dx.doi.org/10.1021/es3003519 | Environ. Sci. Technol. 2012, 46, 8960−8968


Article

Figure 1. Reductive debromination and growth of strain CBDB1 with brominated benzenes as electron acceptor. (A) Formation of benzene from various bromobenzenes. (B) Growth of strain CBDB1 with bromobenzenes as electron acceptor. Symbols: (●) MBB, (○) 1,2-DBB, (▼) 1,3-DBB, (Δ) 1,4-DBB, (■) 1,2,4-TBB. Bromobenzenes were refed to a concentration of 50 μM on day 20 (arrow). Results were obtained from cultures grown in a second transfer with DBB or MBB and a fifth transfer with 1,2,4-TBB. Mean values and standard deviations were calculated from three parallel cultures.

(100% acetonitrile) and a nano-UPLC system (nanoAquity, Waters). Peptides were eluted over the first 90 min with a 6 to 20% solvent B gradient continuing with a gradient over 60 min from 20 to 85% solvent B. Coupling of the nanoUPLC system to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) was performed with TriVersa NanoMate LC-coupler (Advion). Continuous scanning of eluted peptide ions was carried out between 300 and 1600 m/z, automatically switching to MS/MS CID mode (normalized collision energy 35, activation time 30 ms) on top 6 peptide ions exceeding an intensity of 3000. Dynamic exclusion was enabled with an exclusion mass width of ±4 ppm over 3 min. Data were evaluated with tandem mass spectrometry ion search algorithms from the Mascot house server (v2.2.1) (27). The taxonomy ID 255470 of NCBInr (National Center for Biotechnology Information, Rockville, MD) was selected for the search together with tryptic cleavage and a maximum of two missed cleavage sites. Carbamidomethylation at cysteines was given as fixed and oxidation of methionines as a variable modification. As a peptide tolerance a threshold of ±10 ppm was chosen and the MS/MS tolerance threshold was set at ±0.8 Da. Peptides were considered to be identified by Mascot when a false-positive probability of 0.05 (probability-based ion score threshold of 40) was obtained. From identified peptides the protein score and the emPAI (exponentially modified protein abundance index)33 were calculated by Mascot for each identified protein. A higher protein score corresponds to a more confident match between the ion scores and the amino acid sequence of the protein. The emPAI allows an approximate quantitation of the protein by dividing the number of observed peptides by the number of expected peptides followed by an exponential modification of the obtained value.

the reaction mixture was extracted with hexane, and extracts were analyzed for dehalogenation products by gas chromatography as described above. The microtiter plate format assay was set up in a 96 roundbottom well microtiter glass plate (Zinsser Analytic, Frankfurt, Germany). The microtiter plate reader was operated in the anaerobic chamber. Absorbance was measured at 578 nm (methyl viologen absorbance maximum) and 450 nm (background absorbance caused by a condensate formed on the plastic film). Initial absorbance of the assay solution at 578 nm was adjusted to 1.5−3.5 with Ti(III) citrate. Two hundred μL of assay solution were filled into each well and the reaction was started by adding 20 or 40 μL of whole cell suspension containing at least 1 × 107 cells mL−1. After sealing the glass plate with a transparent plastic film (VWR, Dresden, Germany) absorbance was monitored for 15 h at 30 °C. Each plate contained the samples with brominated benzenes as electron acceptor, negative controls without electron acceptor and a positive control with 1,2,3-TCB as electron acceptor, each in six parallels. For all culture determinations three independent parallel cultures were measured. The specific activity was calculated from the slope of the linear part of the measured curves. NanoLC-ESI-MS/MS (LTQ-Orbitrap) Analysis. Strain CBDB1 cultures for Rdh protein analysis contained 2 - 4 × 107 cells mL−1 and were grown under strictly anaerobic conditions with either MBB, one of the three DBB congeners or 1,2,4-TBB. Thirty mL were harvested from each culture by filtration through a 0.2 μm filter. Obtained cells were processed and analyzed as described previously.32 Briefly, cells were lyzed, proteins were digested with trypsin and acquired peptides were purified and concentrated. Peptides were reconstituted in 0.1% formic acid for nanoLC-ESI-MS/MS measurements. Trapping of the samples was done with water containing 0.1% formic acid at flow rates of 15 μL min−1 (nanoAcquity UPLC column, C18, 180 μm × 2 cm, 5 μm, Waters, Eschborn, Germany). After 8 min, the peptides were eluted onto the separation column (nanoAcquity UPLC column, C18, 75 μm × 100 mm, 1.7 μm, Waters, Eschborn, Germany). Chromatography was performed by using 0.1% formic acid in solvents A (100% water) and B

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RESULTS Growth with Brominated Benzenes. D. mccartyi strain CBDB1 reductively debrominated 1,2,4-TBB, 1,2-DBB, 1,3DBB, 1,4-DBB and MBB. The strain was transferred five times with 1,2,4-TBB and two times with DBB or MBB in synthetic medium with either of these brominated benzenes as sole electron acceptor without losing dehalogenating activity. The

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Table 1. Growth Yields and Specific Dehalogenation Rates of Strain CBDB1 when Grown on Different Halogenated Compounds Including the Brominated Benzenes As Electron Acceptora electron acceptor MBB 1,2-DBB 1,3-DBB 1,4-DBB 1,2,4-TBB 2,3-DCP HCB/PeCB PCE/TCE

growth yield [cells mol−1 halogen ion released] 2.9 × 1014 (±3.9 × 1012) 2.5 × 1014 (±2.4 × 1013) 1.9 × 1014 (±9.4 × 1013) 2.0 × 1014 (±2.4 × 1013) 1.8 × 1014 (±6.3 × 1013) 0.8 × 1014 0.2 × 1014/ 0.3 × 1014 2.5 × 1014/ 1.3 × 1014

specific dehalogenation rate [μmol halogen ion day−1 cell−1] 6.2 × 10−11 (±1.2 × 10−12) 7.0 × 10−11 (±7.1 × 10−12) 9.5 × 10−11 (±4.1 × 10−11) 8.8 × 10−11 (±1.1 × 10−11) 9.6 × 10−11 (±5.4 × 10−11) 2.0 × 10−9 nd nd

dehalogenation products

literature

benzene

this study

MBB→benzene

this study

MBB→benzene

this study

MBB→benzene

this study

1,3-DBB/1,4-DBB→ MBB→Benzene

this study

3-MCP 1,2,4,5-TeCB; 1,2,3,5-TeCB; 1,2,4-TCB, 1,3,5-TCB, 1,3-DCB, 1,4-DCB cisDCE, transDCE

23 31 29

a

Abbreviations: TBB - tribromobenzene, DBB - dibromobenzene, MBB - monobromobenzene, DCP - dichlorophenol, MCP - monochlorophenol, HCB - hexachlorobenzene, PeCB - pentachlorobenzene, TeCB - tetrachlorobenzene, TCB - trichlorobenzene, DCB - dichlorobenzene, PCE perchloroethene, TCE - trichloroethene, DCE - dichloroethene, nd - not determined.

107 cells mL−1. Cell numbers increased constantly from day 12 until the end of cultivation. The observed decrease in cell numbers after inoculation (Figure 1B) could be due to contaminations with traces of oxygen introduced during inoculation or shearing forces occurring during the inoculation process or toxic effects of the electron acceptors used. Maximum cell numbers were reached at the end of data acquisition at day 57. An often described uncoupling of dehalogenation and cell growth23 was not observed. From the measured dehalogenation and growth data, molar cell yields of 1.8 × 1014 to 2.8 × 1014 cells per mol bromide released and specific dehalogenation rates of 6.1 × 10−11 to 9.6 × 10−11 μmol bromide day−1 cell−1 were calculated (Table 1). Resting Cell Enzymatic Activity Assay in a Microtiter Plate Format. The photometric activity assay for reductive dehalogenases was set up in a 96 well glass microtiter plate with ten times higher concentrations of electron acceptors than described previously.30 The use of glass instead of polystyrene microtiter plates was essential to avoid adsorption of halogenated compounds to the plate material. Negative controls containing all assay components, an electron acceptor but no cells showed nearly stable methyl viologen absorption values (Figure 2). However, negative controls with cells but without electron acceptor revealed a background activity of methyl viologen oxidation that amounted to about half the rate of methyl viologen oxidation by positive controls supplemented with 1,2,3-TCB and fluctuated around this value for different cultures used (Figure 2). When only one culture was applied in the assay, background activity measured in negative controls was reproducible and stable over all wells. The background activity was not inhibited by 1 mM propyl or ethyl iodide, described to inhibit the corrinoid cofactor containing chlorobenzene reductive dehalogenase CbrA.30 GC-analysis confirmed that electron acceptors were not carried over from the inoculating cultures to the assay via the inoculum. Rotary evaporation to concentrate cells removed such traces. This demonstrated that the inoculum itself was able to take up electrons at a certain rate. Hence, for all calculations the background oxidation of methyl viologen was subtracted from the total methyl viologen oxidation to obtain methyl viologen oxidation mediated by halogenated benzenes. When several

sole final product in all cultures was benzene. Product formation from 1,2,4-TBB, one of the DBB congeners or MBB was observed within 6 days after inoculation (Figure 1A). Within 20 days of incubation 20−40 μM of benzene were formed in all cultures except for cultures supplemented with MBB, and an additional dose of 50 μM was added to all including the MBB cultures. Debromination rates increased and were accompanied by increasing concentrations of benzene in all cultures until day 45 (Figure 1A). From day 45 benzene concentrations stagnated or even decreased and standard deviation of benzene concentrations increased over time (Figure 1A). Benzene is highly volatile and was partially lost through the pierced Teflon-lined rubber septa, especially after a number of sampling points, as observed for 1,2- and 1,4-DBB fed cultures from day 45 to day 57 (Figure 1A). Determination of electron acceptor concentrations in these cultures showed that cultures initially amended with 1,2-DBB and 1,4-DBB still had the dibrominated electron acceptor (18 and 5 μM, respectively) at day 45 but all dibrominated electron acceptor was used up at day 57. During the dehalogenation of 1,2,4-TBB traces of 1,3-DBB, 1,4-DBB and MBB were transiently formed. In all DBB debrominating cultures MBB was detected as an intermediate. The initial rate of debromination was lower in cultures with MBB than in cultures with higher brominated benzenes. In a first passage using a 1,2,4-TBB grown culture as inoculum a lag phase of 36 days was observed (data not shown). In the second passage, traces of benzene were already detected after 6 days, however, within the first 20 days of cultivation the dehalogenation rate of MBB (0.3 μM d−1) was lower than the dehalogenation rates of 1,2,4-TBB or the three DBB congeners (1.1−2 μM d−1) (Figure 1A). Control cultures without inoculum did not form dehalogenation products from any of the supplemented electron acceptors. Transformation of brominated benzenes was accompanied by an increase in cell numbers of strain CBDB1 (Figure 1B). In cultures of the second transfer, cells grew from initial cell numbers of 5 × 105 to 5 × 106 to a final cell number of around 4 × 107 cells mL−1 when 1,2,4-TBB or dibrominated benzenes were added as electron acceptors. The final cell number in cultures supplemented with MBB was slightly lower with 1.9 × 8963



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to MBB was rarely observed. All three dibrominated benzenes were dehalogenated to MBB (Table 2). The activities per mg protein determined by the GC/FID based assay were comparable to activities calculated from the photometric assay and ranged from 2.5 nkat mg−1 for 1,4-DBB to 10.4 nkat mg−1 for 1,2-DBB. MBB was not dehalogenated in the GC/FID-based activity assay. In the next step, the photometric activity test was applied to investigate induction of reductive dehalogenases during cultivation with bromobenzenes. For all bromobenzene grown cultures, activities were determined with the brominated electron acceptor on which the culture was grown. For comparison, the activity with all bromobenzenes was measured for a 1,2,3-TCB grown culture (Figure 3). No improved activity

Figure 2. Photometric monitoring of methyl viologen oxidation at 578 nm. 1,2,3-TCB was used as an electron acceptor. Whole cells showed a background activity even without electron acceptor that was subtracted from calculated activities. The arrow represents the time period used for slope calculation within the linear part of the curves (a).

cultures were tested in one microtiter plate assay, separate negative controls for each culture were included. When cell suspensions of D. mccartyi strain CBDB1 cultivated on 1,2,4-TBB were used as a catalyst, dehalogenation of 1,2,3-TCB, 1,2,4-TBB, 1,2-DBB, 1,3-DBB, and 1,4-DBB was observed (Table 2). The highest activities per mg cell protein were calculated for compounds possessing doubly or singly flanked halogen substituents such as 1,2,4-TBB (13.2 nkat mg−1), 1,2-DBB (12.4 nkat mg−1) and 1,2,3-TCB (5.9 nkat mg−1). The lowest activity per mg cell protein was determined for 1,4-DBB (2.8 nkat mg−1). Although debromination of MBB was observed during cultivation of strain CBDB1, no activity was observed in the activity assays (Table 2). Also no MBB debromination activity was detected with ethyl viologen, an electron donor with more negative redox potential than methyl viologen, or when more cells were added to the test. Parallel to each photometric activity assay, cell suspensions were used for a GC/FID-based activity assay as described previously.30 In this assay, 1,4-DBB was detected as the first dehalogenation product of 1,2,4-TBB. Further dehalogenation

Figure 3. Specific activities of whole cell suspensions expressed in nkat per mg of total protein measured with different electron acceptors. Tested cultures of strain CBDB1 were either cultivated on 1,2,3-TCB (gray bars) or on the same e−-acceptor used for the activity assay (black bars).

was detected in cultures grown with bromobenzenes compared to cells cultivated with 1,2,3-TCB (Figure 3). Even higher

Table 2. Activities of D. mccartyi Strains Using Different Halogenated Compounds As Electron Acceptors. Standard Deviations Are Deduced from One Run on a Microtiter Plate with One Culturea photometric assay

gas chromatographic assay

e−-acceptor for cultivation

e−-acceptor for assay

sp. act. [nkat mg protein−1]

sp. act. [nkat mg protein−1]

dehalogenation products

1,2,4-TBB 1,2,4-TBB 1,2,4-TBB 1,2,4-TBB 1,2,4-TBB 1,2,4-TBB 1,2,3-TCB and 1,2,4-TCB 1,2,3-TCB and 1,2,4-TCB 1,2,3-TCB and 1,2,4-TCB HCB PeCB PCE PCE

MBBb 1,2-DBBb 1,3-DBBb 1,4-DBBb 1,2,4-TBBb 1,2,3-TCBb 1,2,3-TCB 1,2,4-TCBb PeCBb HCBb PeCBb PCEc PCEd

0 12.4 ± 0.7 5.6 ± 0.3 2.8 ± 0.9 13.2 ± 2.1 5.9 ± 0.3 nd nd nd nd nd nd nd

0 10.4 7.3 2.5 10.2 5.5 11 ± 0.7 0.3 ± 0.0 171 ± 12 3.36 22 0.42 ± 0.15 4.5

none MBB MBB MBB 1,4- and 1,3-DBB 1,3-DCB 1,3-DCB 1,3- and 1,4-DCB 1,2,3,5- and 1,2,4,5-TeCB 1,3,5-TCB, 1,4-and 1,3-DCB 1,3,5-TCB, 1,4-and 1,3-DCB cisDCE/VC TCE/VC

literature this this this this this this 30 30 30 31 31 52 43

study study study study study study

a

Abbreviations: sp. act. - specific activity, TBB - tribromobenzene, MBB - monobromobenzene, DBB - dibromobenzene, TCB - trichlorobenzene, PeCB - pentachlorobenzene, HCB - hexachlorobenzene, PCE - tetrachloroethene, TCE - trichloroethene, cisDCE - dichloroethene, VC - vinyl chloride, nd - not determined. bD. mccartyi strain CBDB1, whole cells. cD. mccartyi strain 195, whole cells. dD. mccartyi strain 195, cell membrane fraction. 8964



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Table 3. Reductive Dehalogenase Homologous (Rdh) Proteins Identified in Cultures Grown with Brominated Benzenesa e−-acceptor

MBB

1,2-DBB

1,3-DBB

1,4-DBB

1,2,4-TBB

RdhCBDB1

Score

emPAI

Score

emPAI

Score

emPAI

Score

emPAI

Score

emPAI

CbdbA80 CbdbA84 (CbrA)b CbdbA88 CbdbA1453 CbdbA1455 CbdbA1618 CbdbA1638

221

0.75

614

2.44

461 306

1.60 0.51

1147 127

3.82 0.34

523 379

1.75 0.60

61 128 121

0.25 0.13 0.19

387 314 195 208

0.52 0.60 0.28 0.43

254 107

0.51 0.13

279 293 111

0.44 0.60 0.13

85 151 92

0.19 0.21 0.20

a

Abbreviations: score - mascot protein score; probability value for the identification of a protein; higher protein scores correspond to a more confident match between the ion scores and the amino acid sequence of the protein; emPAI - exponentially modified protein abundance index. A higher emPAI indicates a higher abundance of an Rdh protein. Protein score and emPAI were calculated from ion scores that exceeded the significance threshold (see Material and Methods). bPutative chlorobenzene reductive dehalogenase.

activities per mg cell protein were calculated for cell suspension grown with 1,2,3-TCB and using 1,2-DBB and 1,2,4-TBB as electron acceptor for the activity assay (Figure 3). Similar specific activities were observed for 1,3-DBB and 1,4-DBB after cultivation of the cells with 1,2,3-TCB or the respective bromobenzene. Protein Expression Analysis. Cultures grown with bromobenzenes were analyzed for presence of Rdh proteins using a shotgun proteomics approach. Overall, seven distinct Rdh proteins were identified in the protein extracts of cultures with the tested brominated benzenes, although only a subset of 4−6 of these were identified in any one culture condition (Table 3). CbdbA80 was the Rdh protein with the highest abundance in all tested samples according to the emPAI-value (exponentially modified protein abundance index), which was used in this study to estimate the approximate relative abundance of Rdh proteins.33 Expression of the trichlorobenzene reductive dehalogenase gene (cbrA, cbdbA84) was observed after growth of strain CBDB1 with 1,2,4-TBB, 1,4DBB, or 1,3-DBB. Rdh protein CbdbA1455 was detected in all tested samples. In cultures that expressed the reductive dehalogenase gene cbdbA84, abundance of Rdh protein CbdbA1455 was at a same or even higher level compared to CbdbA84.

Halogen substituents on aromatic compounds have previously been classified as doubly flanked, singly flanked or isolated when they have two, one or none neighboring halogen substituents, respectively.37 In strain CBDB1 the dehalogenation pathway from 1,2,4-TBB follows the reactions of the dehalogenation of 1,2,4-TCB.19,31 Strain CBDB1 cultures incubated with 1,2,4-TBB removed preferentially the singly flanked bromine substituent at position 2, resulting in a 5-fold higher formation of 1,4-DBB than 1,3-DBB. This demonstrates that the reaction specificities of the catalyzing enzyme systems are similar. Isolated substituents were removed in bromobenzenes (1,3-DBB, 1,4-DBB, MBB) but not in chlorobenzenes.19 The microtiter plate format photometric activity assay confirmed the advantages of photometric assays as a real-time tool to monitor enzymatic reactions. Many compounds can be tested in parallel by this screening method, avoiding tedious cultivation experiments. Additionally, the test enables a first assessment independent from specific analytical detection procedures for the different electron acceptors used and could potentially also be used for enrichment cultures. It would then allow for the quick assessment of the remediation potential of dehalogenating microcosms from contaminated field sites and the cultivation independent investigation of new electron acceptors of uncharacterized dehalogenating bacteria. However, there are factors which limit the applicability of the test, especially when less defined or less active samples are used. For example, precipitates or soluble oxidizing components can function as electron acceptor or catalyst or both and cause high background activity. Also the test with whole cells will only detect those reductive dehalogenases that are located on the surface of a cell. Both limitations would make further purification procedures and/or cell lysis necessary. In general, the activity assays depicted accurately the dehalogenating potential and the catalyzed pathways. However, although MBB was dehalogenated to benzene in cultures, reductive dehalogenation of MBB was neither observed in the GC-based nor in the photometric activity assays. This could be due to the fact that the tests are less sensitive with MBB as electron acceptor or that the tests in the current form do not work at all for the MBB reductive dehalogenase. Increasing the sensitivity by using more cells, by using an electron donor with more negative redox potential or by increasing the incubation time did not lead to detectable benzene formation and therefore did not resolve this question. Factors that could influence the ability to form benzene from MBB in the activity test include very high oxygen sensitivity of the MBB

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DISCUSSION Here we describe the stoichiometric formation of benzene by D. mccartyi strain CBDB1 during cultivation with 1,2,4-TBB, 1,2-DBB, 1,3-DBB, 1,4-DBB, or MBB as electron acceptor. Such a removal of all halogen substituents from a contaminant is an important step for an efficient detoxification and mineralization. The microbially mediated removal of all halogen substituents from chlorinated aliphatics has been shown for several pure Dehalococcoides strains.22,34,35 In D. mccartyi strain 195 enzymatic activity was detected that completely dehalogenated brominated ethanes, pentenes and propenes36 but no growth with the brominated compounds was demonstrated. Our results demonstrate that strain CBDB1 grows with brominated benzenes as sole terminal electron acceptors, indicating that bromobenzenes are used as electron acceptors in a respiratory process. With all tested bromobenzenes growth of CBDB1 was observed over several transfers. The observed growth yields of 1.8 × 1014 to 2.9 × 1014 cells per mole of bromide released are in good accordance with growth yields of Dehalococcoides grown with polybrominated biphenylethers,27 chlorophenols23 or tetra- and trichloroethene.29 8965



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that brominated and chlorinated compounds are reduced by the same Rdh protein and that strain CBDB1 has an even wider dehalogenation potential for brominated benzenes than for chlorinated benzenes. The capability to use naturally occurring brominated organic compounds that are common in marine sediments48,49 could, in part, explain the widespread occurrence of Dehalococcoides related 16S rRNA 50 and reductive dehalogenase homologous genes51 in marine subsurface sediments.

dehalogenating activity and the need for even stronger electron donors. The specific activities of whole cells of strain CBDB1 were higher for brominated benzenes than for the respective chlorinated benzenes. For example, whereas here we found a value of 13.2 nkat mg−1 for 1,2,4-TBB dehalogenation, the dechlorination of 1,2,4-TCB was only at 0.3 nkat mg−1.30 Higher specific activity toward bromine substituents in an in vitro activity assay was also observed previously.36 The purified trichloroethene reductive dehalogenase transformed 1,2dibromoethane to ethene at a specific activity of 500 nkat mg−1 while 1,2-dichloroethane was dechlorinated at 125 nkat mg−1. Vinyl bromide and vinyl chloride were both reduced to ethene at specific activities of 3 and 0.6 nkat mg−1, respectively. The observation of brominated compounds being dehalogenated faster than chlorinated compounds in biological reactions is in agreement with studies on abiotic chemical dehalogenations in which the aryl-bromine bond was shown to be weaker than the aryl-chlorine bond.38,39 The microtiter plate format activity assay was also used for the analysis of rdh gene expression under different cultivation conditions. Several studies have shown that specific reductive dehalogenase genes are induced under specific cultivation conditions.40,41 Analysis of the genome of strain CBDB1 revealed that almost all rdh genes are linked to regulatory genes suggesting tight transcriptional control of their expression.42 Although a large number of rdh gene sequences have been found in pure and mixed cultures by molecular approaches, little is known about the function of the putative reductive dehalogenases encoded by these genes and the reason for their variety. Until now, tetrachloroethene and trichloroethene dehalogenases,36,43 vinyl chloride dehalogenases34,44 and a trichlorobenzene dehalogenase45 have been identified in Dehalococcoides species. The trichloroethene Rdh protein dehalogenates chlorinated and brominated aliphatic compounds.36 So far, no enzymes have been identified that specifically transform brominated aromatics. Results from our activity experiments using cells pregrown with either brominated or chlorinated benzenes provided us with evidence that the same enzymes are likely responsible for the dehalogenation of both bromobenzenes and chlorobenzenes. For instance, our activity assays with 1,3- and 1,4-DBB revealed similar specific activities for cells pregrown with brominated benzenes or 1,2,3-TCB. Additionally, even higher specific activities were obtained for 1,2-DBB and 1,2,4-TBB when cells were pregrown on 1,2,3-TCB instead of the respective brominated compound. According to emPAI-values obtained from mass spectrometry, the most abundant Rdh protein in all tested samples was CbdbA80, which previously was shown to be expressed by strain CBDB1 after cultivation with a mixture of 1,2,3-TCB and 1,2,4-TCB45 and after cultivation with 2,3-dichlorophenol.46 Also CbdbA84, the trichlorobenzene reductive dehalogenase of strain CBDB1,45 was frequently detected in cultures grown on bromobenzenes. In total, seven Rdh proteins were detected using shotgun proteomics, which is in good accordance with results obtained during transcription analysis of different Dehalococcoides strains showing the simultaneous expression of multiple rdh genes.40,41,47 While some of the Rdh proteins seem to be constitutively expressed (e.g., CbdbA80), others were induced, indicating their involvement in reductive dehalogenation of trichlorobenzenes, tribromo-, dibromo- and monobromobenzene. In summary, our study provides evidence

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

A.W. and M.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Bernd Krostitz for technical support. We are also grateful to Kenneth Wasmund for helpful discussions. This work was supported by the European Research Council (ERC Microflex project, No. 202903-2), and the Deutsche Forschungsgemeinschaft (FOR1530).

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Growth of Dehalococcoides mccartyi strain CBDB1 by reductive

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