Anaerobic Dehalogenation of Chloroanilines by Dehalococcoides

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Anaerobic Dehalogenation of Chloroanilines by Dehalococcoides mccartyi Strain CBDB1 and Dehalobacter Strain 14DCB1 via Different Pathways as Related to Molecular Electronic Structure Shangwei Zhang, Dominik Wondrousch, Myriel Cooper, Stephen H. Zinder, Gerrit Schüürmann, and Lorenz Adrian Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05730 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Environmental Science & Technology

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Anaerobic Dehalogenation of Chloroanilines by

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Dehalococcoides mccartyi Strain CBDB1 and Dehalobacter

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Strain 14DCB1 via Different Pathways as Related to

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Molecular Electronic Structure

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Shangwei Zhang,†‡§ Dominik Wondrousch,†§ Myriel Cooper,‡ Stephen H. Zinderǁ,

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Gerrit Schüürmann⃰ †§ and Lorenz Adrian ⃰ ‡┴

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†UFZ Department of Ecological Chemistry, Helmholtz Centre for Environmental Research,

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Permoserstraße 15, 04318 Leipzig, Germany

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‡UFZ Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental

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Research, Permoserstraße 15, 04318 Leipzig, Germany

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§Technical University Bergakademie Freiberg, Institute for Organic Chemistry,

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Leipziger Straße 29, 09596 Freiberg, Germany

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┴Technische Universität Berlin, Fachgebiet Applied Biochemistry,

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Gustav-Meyer-Allee 25, 13355 Berlin, Germany

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ǁ

Department of Microbiology, Wing Hall, Cornell University,

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Ithaca, New York 14853, United States

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*Corresponding authors: Gerrit Schüürmann, Tel +49-341-235-1262, Fax +49-341-235-45-

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1262, Email [email protected], and Lorenz Adrian, Tel +49-341-235-1435, Fax +49-

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341-235-1443, Email [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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Dehalococcoides mccartyi strain CBDB1 and Dehalobacter strain 14DCB1 are or-

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ganohalide-respiring microbes of the phyla Chloroflexi and Firmicutes, respectively.

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Here we report the transformation of chloroanilines by these two bacterial strains via

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dissimilar dehalogenation pathways, and discuss the underlying mechanism with

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quantum chemically calculated net atomic charges of the substrate Cl, H and C at-

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oms. Strain CBDB1 preferentially removed Cl doubly flanked by two Cl or by one Cl

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and NH2, whereas strain 14DCB1 preferentially dechlorinated Cl that has an ortho H.

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For the CBDB1-mediated dechlorination, comparative analysis with Hirshfeld charges

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shows that the least negative Cl discriminates active from non-active substrates in 14

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out of 15 cases, and may represent the preferred site of primary attack through

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cob(I)alamin. For the latter trend, 3 of 7 active substrates provide strong evidence,

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with partial support from 3 of the remaining 4 substrates. Regarding strain 14DCB1,

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the most positive carbon-attached H atom discriminates active from non-active

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chloroanilines in again 14 out of 15 cases. Here, regioselectivity is governed for 10 of

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the 11 active substrates by the most positive H attached to the highest-charge (most

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positive or least negative) aromatic C carrying the Cl to be removed. These findings

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suggest the aromatic ring H as primary site of attack through the supernucleophile

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Co(I), converting an initial H-bond to a full electron transfer as start of the reductive

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dehalogenation. For both mechanisms, one- and two-electron transfer to Cl (strain

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CBDB1) or H (strain 14DCB1) are compatible with the presently available data.

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Computational chemistry research into reaction intermediates and pathways may fur-

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ther add in understanding the bacterial reductive dehalogenation at the molecular

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level.

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INTRODUCTION

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Chloroanilines are intermediates in the synthesis of dyes, plastics, pharma-

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ceuticals and pesticides.1, 2 Aromatic pesticides with amino groups such as diuron,

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linuron, anilide, propanil, and triclocarban can be transformed to chloroanilines in na-

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tural environments.3,

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corresponding chloroanilines with the fungicide pentachloronitribenzene being a pro-

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minent example, which contributes to the widespread occurrence and accumulation

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of chloroanilines in groundwater, soil, sludge, household waste, rainwater and crops.2

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Chloroanilines may enter the food chain and therefore represent a potential health

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risk for animals and humans. Some chloroaniline congeners are recognized as neph-

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rotoxic, cytotoxic or carcinogenic pollutants5, 6 and may bind to crops, yielding non-

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extractable residues.7 Adsorption to metal oxide surfaces such as birnessite (MnO2)

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and iron(III) oxide may trigger chloroanilino radical formation through electron trans-

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fer, resulting in various dimerization products. 8

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Moreover, chlorinated nitrobenzenes can be reduced to the

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Whereas in oxic environments lower chlorinated anilines can be transformed

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to chlorocatechols and subsequently used as carbon and nitrogen source by a va-

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riety of microorganisms,9, 10 little is known about their transformation in anoxic envi-

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ronments. Anaerobic transformation was observed in methanogenic aquifers and

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aquifer slurries,1, 11-13 flooded soils14 and estuarine sediments;15 the responsible orga-

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nisms, however, were not identified. In mixed consortia, different dehalogenation

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pathways were observed: In anaerobic enrichment cultures with estuarine sediments

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as an inoculum, pentachloroaniline (PeCA) was converted to 2,3,4,5-tetrachloroani-

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line (2,3,4,5-TeCA),15 i.e. via ortho-dechlorination. A methanogenic enrichment cul-

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ture transformed PeCA to mainly 2,3,5,6-TeCA with minor amounts of 2,3,4,5-TeCA

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via dehalogenation of the chlorine substituents in para- and ortho-position.14, 15


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Recently, the transformation of 2,3-dichloroaniline (2,3-DCA) to 3-monochloro-

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aniline (3-MCA) by Dehalococcoides mccartyi strain CBDB1 was reported.16 Mem-

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bers of the genus Dehalococcoides such as strain CBDB1 depend on organohalides

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as terminal electron acceptor for respiration, energy production and growth. Dehalo-

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coccoides can transform a large range of different organohalides, and thus are con-

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sidered as key organisms for natural attenuation and bioremediation of halogenated

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xenobiotics.17 Besides 2,3-DCA, Dehalococcoides strain CBDB1 has been shown to

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transform halogenated ethenes, benzenes, phenols, dioxins, benzonitriles and biphe-

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nyls.16 However, strain CBDB1 was not able to transform 2,4- and 2,6-DCA. Reduct-

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ive dehalogenation of higher chlorinated anilines has not been investigated with

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strain CBDB1 or other pure strains. When using 2,3-DCA as terminal electron accep-

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tor, strain CBDB1 dehalogenated the chlorine substituent in ortho-position of the ami-

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no group, but abstraction of the chlorine in meta-position was not observed.16 A simi-

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lar dehalogenation pattern was observed for 1,2,3-trichlorobenzene. Strain CBDB1

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dehalogenated the doubly flanked chlorine, whereas the singly flanked chlorines

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were not removed,18 indicating that the amino group in chloroanilines supports deha-

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logenation in a similar manner as the chlorine substituents in chlorobenzenes. Elec-

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tron density based natural population analysis (NPA) revealed that the most positive

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halogen partial charge could predict with a success rate of 96% the regioselective

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dehalogenation by strain CBDB1 in different compound classes.16 However, chloro-

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anilines were the few exceptions whose dehalogenation pathway was not predicted

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correctly using NPA and Mulliken charges. In addition, the prediction of dehalogena-

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tion pathways based on the most positive halogen partial charge was not applicable

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to organohalide-respiring bacterial strains from bacterial classes other than the Deha-

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lococcoides.



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Like Dehalococcoides strains, Dehalobacter strains are specialized on respira-

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tion with organohalides as terminal electron acceptors and cannot grow by fermenta-

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tion.19, 20 Strains of both genera use hydrogen as electron donor. The enzymes that

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catalyze the dehalogenation step in both genera are called reductive dehalogenases.

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They contain two iron sulfur clusters and a corrinoid ring in the active site, show sig-

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nificant phylogenic relatedness,20 and thus seem to be very similar.21 Nevertheless,

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very different electron acceptor ranges and catalyzed reaction pathways were ob-

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served.20 Pure cultures, defined co-cultures or enrichments containing Dehalobacter

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spp. have been described to reductively dehalogenate chlorinated methanes,

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ethanes, ethenes, cyclohexanes, benzenes, phenols, biphenyls and phthalides.19, 20,

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22, 23

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transform singly flanked or isolated chlorine substituents. For instance, Dehalobacter

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strains 12DCB1, 13DCB1 and 14DCB1 dehalogenated 1,2-dichlorobenzene, 1,3-di-

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chlorobenzene and 1,4-dichlorobenzene, respectively, yielding monochlorobenzene

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or even some benzene.19 Therefore, Dehalobacter and Dehalococcoides spp. are

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complementary regarding organohalogen transformation. The potential for reductive

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dehalogenation of chlorinated anilines by members of the genus Dehalobacter has

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not been explored.

In addition, several Dehalobacter strains have been described to preferentially

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While dehalogenation may render organohalides less toxic and more suscepti-

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ble for subsequent mineralization under aerobic conditions, reductive dehalogenation

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may also result in the accumulation of more toxic compounds.24 The deeper under-

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standing of dehalogenation pathways and mechanisms of different dehalogenating

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strains may allow the enhanced fate prediction of organohalogens from anthropoge-

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nic sources in the environment and a targeted application of such organisms in bio-

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stimulation and bio-augmentation processes.


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In this study, two representative bacterial species, Dehalococcoides mccartyi

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CBDB1 and Dehalobacter sp. 14DCB1 with different dehalogenation pathways des-

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cribed for chlorobenzenes,18, 19, 25 were selected to investigate the anaerobic transfor-

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mation of chlorinated anilines. The objectives of this study were (1) to investigate the

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pathways and features of dehalogenation of chloroanilines by both stains via cultiva-

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tion and whole-cell enzymatic activity tests, and (2) to explore the dehalogenation

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mechanisms through complementary computational chemistry analyses of the elec-

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tron-acceptor substrates.

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MATERIALS AND METHODS

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Chemicals. Aniline (C6H5NH2) and fifteen of the nineteen possible chloroani-

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line congeners were obtained from ABCR GmbH, Alfa-Aesar, or Merck Sigma-Aldrich

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at the highest available purity (Table S1, Supporting Information). The trichloroani-

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lines 2,3,5-TrCA, 2,3,6-TrCA and the tetrachloroanilines 2,3,4,5-TeCA, 2,3,4,6-TeCA

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were not available for the experiments, but included in the electron density calcula-

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tions.

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Cultivation of Dehalococcoides mccartyi Strain CBDB1 and Dehalobac-

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ter Strain 14DCB1. The two strains were cultivated as described previously with mi-

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nor modification.19, 24 Briefly, both strains were grown in vitamin amended, titanium

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(III) citrate reduced, bicarbonate-buffered mineral medium with 5 mM acetate as car-

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bon source and hydrogen as electron donor (7.5 mM nominal concentration). All 15

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chloroanilines were individually used for cultivation of strain CBDB1 and seven (2,3-

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DCA, 3,4-DCA, 2,3,4-TrCA, 2,4,5-TrCA, 3,4,5-TrCA, 2,3,5,6-TeCA and PeCA) for

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strain 14DCB1. Chloroanilines were added to the medium at concentrations of 40



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µM. The cultures were first pressurized to 0.35 bar overpressure of N2:CO2 mixture

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(80:20%) and then to 0.45 bar overpressure of hydrogen. Both strains were inocu-

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lated to a starting cell number of about 2.0 × 106 cells/mL. Cultures were set up in

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triplicates and were statically incubated in the dark at 30 °C. Cells were quantified by

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direct epifluorescence microscopic cell counting on agarose-coated slides after stain-

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ing with SybrGreen as described previously.26 Chemical controls without cells, nega-

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tive controls without terminal electron acceptors, and positive controls with hexa-

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chlorobenzene, or with a mixture of 1,2,3- and 1,2,4-trichlorobenzene were set up for

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the strains CBDB1 and 14DCB1, respectively. Cultures were re-fed with 40 µM of the

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respective electron acceptor and re-gassed every seven days.

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Enzymatic Activity Assay. A whole-cell enzymatic activity assay using re-

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duced methyl viologen as artificial electron donor and chloroanilines as electron

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acceptor was used to quantify reductive dehalogenation. All fifteen available

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chloroanilines were employed individually for both species. Activity was either as-

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sessed by gas chromatography with flame ionization detection of dehalogenated

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products or by photometric quantification of the decolorization of reduced methyl vio-

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logen during dehalogenation as described previously.27,

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components of reactions mixtures, which contained 732.5 µL anaerobic water, 125

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µL methyl viologen (10 mM), 125 µL potassium acetate buffer (1 M), 5.0 µL titanium

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(III) citrate (15%), and 12.5 µL electron acceptor (50 mM) in 1.0 mL reaction mixture.

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Whole-cell assays were set-up in an anaerobic glove box in 2 ml HPLC crimp glass

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vials with 1.0 mL reaction mixture and 150 µL whole cells from parent cultures.

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Chemical controls and negative controls without electron acceptor were prepared in

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each test. The cell number of Dehalococcoides mccartyi CBDB1 and Dehalobacter

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sp. 14DCB1 in parent cultures used for activity assays were 1.9 × 107 and 1.2 × 107

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cells/mL, respectively. After incubating for 24 h at 30 °C, all samples were sacrificed ACS Paragon Plus Environment

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Here we optimized the

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for analysis of chlorinated anilines by GC-FID and GC-MS.

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Sample Preparation and Analysis. Samples (1.0 mL from cultures, or 1.15

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mL from enzymatic activity assays) were sacrificed at planned sampling points. Each

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sample was mixed with 400 µL hexane and extracted in a shaker with 800 rpm for 24

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h. Then the hexane extract was analyzed with a Hewlett-Packard GC 6890 coupled

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to a flame ionization detector, equipped with an HP-5 capillary column (30 m length,

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0.25 mm i.d., 0.25 µm film thickness). The column temperature was kept at 70 °C for

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1 min, and then increased to 120 °C at 4 °C/min, 126 °C at 2 °C/min, 136 °C at 0.5

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°C/min, 160 °C at 2 °C/min, and 300 °C at 20 °C/min. Nitrogen was used as carrier

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gas at a rate of 1.0 mL/min. Auto injection with 1.0 µL samples was done in the split-

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less mode. The temperature of the injector and detector were 240 and 300 °C,

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respectively. Chloroanilines for which no standards were commercially available were

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analyzed by GC-MS (Agilent 7890A GC-5975 MSD) with an HP-5 column (30 m

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length, 0.25 mm i.d., 0.25 µm film thickness). The initial temperature of 70 °C was

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held for 1 min, after which the temperature was increased to 100 °C at 10 °C/min,

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and finally raised to 200 °C at 4 °C/min. Helium was used as carrier gas with a rate of

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1.0 mL/min. Auto injection with 1.0 µL samples was done in splitless mode. The

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temperatures of the injector, MSD source, quadrupole, and transfer line were 280,

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230, 150 and 280 °C, respectively.

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Computational Details. Quantum chemical calculations including optimiza-

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tion of molecular geometries were performed at the B3LYP and BLYP density func-

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tional theory (DFT) level of theory employing the basis sets Def2-SVP and Def2-

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TZVP as implemented in Gaussian 09 Revision C.01.29 All ground-state geometries

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were verified through the absence of imaginary frequencies of the associated Hes-

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sian matrices. Besides gas-phase calculation, aqueous solution was simulated



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through the COSMO30 continuum-solvation model augmented by non-electrostatic

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contributions as implemented in the CPCM module of Gaussian. Net atomic charges

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were quantified using the Hirshfeld method31 through Multiwfn 3.3.8,32 and by em-

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ploying natural population analysis (NPA) of the natural bond orbital (NBO) scheme33

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calculated with NBO 5.9.34

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RESULTS

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Biotransformation of Chloroanilines by Bacterial Strains CBDB1 and

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14DCB1. Both Dehalococcoides mccartyi strain CBDB1 and Dehalobacter sp. strain

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14DCB1 dehalogenated chloroanilines in cultures with hydrogen as electron donor.

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Seven chloroanilines (2,3-DCA, 3,4-DCA, 2,3,4-TrCA, 2,4,5-TrCA, 3,4,5-TrCA,

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2,3,5,6-TeCA and PeCA) were transformed by strain CBDB1 (Figure 1, Figure S1 for

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2,3,5,6-TeCA). All these chloroanilines except PeCA were also dechlorinated in the

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culture of strain 14DCB1, however, with different rates and to different products (Fig-

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ure 2). In negative controls without cells, the concentrations of the added chloroani-

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lines were almost constant over time, and no dehalogenated products were formed.

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With strain CBDB1, the first products were observed after 7 days of incubation

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(Figure 1, Figure S1). Both 2,3-DCA and 3,4-DCA were transformed to 3-MCA, how-

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ever, the transformation of 2,3-DCA was faster than the transformation of 3,4-DCA.

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For TrCAs, chlorine substituents flanked by two other chlorine substituents were

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more reactive than chlorine substituents ortho to one Cl and to the amino group. Still

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slower was the removal of Cl if that was ortho to one other Cl and to H. Taking 2,3,4-

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TCA as an example, the C3-attached Cl was abstracted preferably with 2,4-DCA as

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main product. As a minor reaction, removal of C2-attached Cl occurred yielding 3,4-


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DCA, but no 2,3-DCA was produced, indicating the resistance of C4-attached Cl to

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microbially mediated dehalogenation. Long-term incubations of PeCA yielded 2,4,6-

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TrCA, 2,4,5-TrCA, 2,5-DCA and 3,5-DCA as ultimate metabolites. To identify which

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tetrachloroanilines were formed from PeCA, a CBDB1 culture grown with hexachloro-

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benzene as electron acceptor was incubated for 12 h at 30 °C with PeCA. Product

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analysis with GC-MS yielded two peaks at retention times 18.58 min and 18.76 min

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associated with an m/z value of 230.8 qualifying them as tetrachloroanilines. One

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peak (18.58 min) had almost the same retention time as a neat standard of 2,3,5,6-

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TeCA (18.57 min), but it might also be 2,3,4,5-TeCA for which we could not obtain a

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neat standard. The second peak at 18.76 min was likely to represent 2,3,4,6-TeCA

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as the other tetrachloroaniline. Production of 2,4,6-TrCA from PeCA indicated further

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that 2,3,4,6-TeCA was an intermediate in the transformation of PeCA, because the

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microbial dehalogenation proceeds step by step. No metabolites were found when

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strain CBDB1 was incubated with 2-MCA, 3-MCA, 4-MCA, 2,4-DCA, 2,5-DCA, 2,6-

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DCA or 2,4,6-DCA for 55 days (data not shown). A common characteristic of all

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these non-transformed chlorinated anilines is that they do not contain vicinal chlorine

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substituents.

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Dehalobacter sp. strain 14DCB1 dehalogenated three of the seven tested

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chloroanilines quantitatively (2,3,4-TrCA, 2,4,5-TrCA, 2,3,5,6-TeCA). For three other

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compounds (2,3-DCA, 3,4-DCA, 3,4,5-TrCA) minor amounts of products were formed

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(Figure 2), and PeCA was not transformed. Thus strain 14DCB1 removed Cl only if

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that was ortho to H and to either Cl or NH2. This pattern found for strain 14DCB1 is

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termed lateral dechlorination because only the two Cl substituents at the beginning

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and end of a Cl substituent series are removed. Thus, for 3,4,5-TrCA Cl at C3 and at

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C5 are removed, whereas for 2,3,4-TrCA C2-Cl and C3-Cl without ortho H are not ab-

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stracted. Interestingly, with the isomer 2,4,5-TrCA as substrate both C2-Cl and C4-Cl ACS Paragon Plus Environment


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are cleaved, but C5-Cl resists 14DCB1-mediated dehalogenation. The lack of in-

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crease in dehalogenation rate of some substrates in the last period of incubation

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(Figures 1 and 2) could result from time-dependent variations in chloroaniline toxicity

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or reflect different bacterial growth stages, and may be subject to future investigati-

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ons (day 28 had not been included in sampling).

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As a result of the strain-specific dehalogenation selectivities, the metabolite

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patterns differ between Dehalococcoides mccartyi strain CBDB1 and Dehalobacter

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sp. strain 14DCB1 (Figure 3). Strain CBDB1 only dehalogenated chloroanilines with

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vicinal chlorines, preferring the abstraction of doubly-flanked substituents. By con-

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trast, strain 14DCB1 dehalogenated chloroanilines with at least one unsubstituted

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aromatic carbon, removing Cl ortho to H and one other substituent (Figures 2 and 3).

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Here, the presence of at least one H at a vicinal aromatic carbon is mandatory to

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make the substrate active for dehalogenation. Accordingly, PeCA resists 14DCB1-

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mediated Cl abstraction, which holds also for the earlier demonstrated respective sta-

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bility of hexachlorobenzene.19

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Enzymatic Activity Assessment. An in vitro test was employed using methyl

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viologen as artificial electron donor to measure the whole-cell activity of strains

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CBDB1 and 14DCB1 with the 15 chloroanilines independently from growth, employ-

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ing hexachlorobenzene and 1,2,3-trichlorobenzene as electron acceptors, respect-

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ively (Table 1). The results confirm the dehalogenation patterns observed during cul-

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tivation. Chloroanilines with neighboring Cl atoms were transformed by strain CBDB1

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cells, and Cl flanked by two other Cl was dechlorinated with the highest specific acti-

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vity. By contrast, strain CBDB1 did not dehalogenate chloroanilines without neighbor-

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ing Cl atoms. The aniline group ortho to Cl provided only weak support for CBDB1-

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mediated dechlorination, converting for instance 2,3-DCA to 3-MCA. Deamination


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was never observed.

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As seen in the cultivation, cells of Dehalobacter sp. strain 14DCB1 trans-

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formed chlorinated anilines with at least one unsubstituted aromatic carbon except

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for the three monochloroanilines (Table 1). Moreover, for both bacterial strains the

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metabolites found with the enzymatic activity assay are in line with the pathways ob-

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served during cultivation. For example, 2,3,4-TrCA was dechlorinated to 2,4-DCA

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and 3,4-DCA at a ratio of 9:1 by CBDB1, whereas 2,3,4-TrCA was exclusively trans-

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formed to 2,3-DCA by 14DCB1.

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Hirshfeld Charge vs CBDB1-Mediated Dechlorination. To explore potential

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relationships between the electronic structure of the substrates and their activity for

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dechlorination, Hirshfeld31 net atomic charges have been calculated because of their

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respective suitability in a previous study.16 Figures 4 and S2 show these charges for

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all Cl and associated aromatic C atoms as well as for all H atoms of the 15 experi-

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mentally tested chloroanilines and of four commercially unavailable derivatives; the

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corresponding NPA33 net atomic charges are available in Figure S3. Here, the color

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code employed for the Cl atoms is as follows: red indicates Cl abstraction only

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through strain CBDB1, green Cl abstraction only by strain 14DCB1, and gold Cl abs-

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traction with both bacterial strains.

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Regarding strain CBDB1, detailed inspection of Figure 4 unravels links bet-

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ween the calculated net atomic charge of Cl, qCl, and the experimentally observed

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dechlorination activity that can be summarized in the two following rules, considering

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charges as identical if their difference is less or equal 0.003.

302 303

Strain CBDB1 Dechlorination Rule 1:



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The chloroaniline is active as a strain CBDB1 dehalogenation substrate if there is at

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least one Cl with qCl ≥ –0.042, ignoring qCl differences up to 0.003.

306 307

Strain CBDB1 Dechlorination Rule 2:

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Regarding strain CBDB1 regioselectivity, the least negative Cl atom is cleaved pre-

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ferably, again ignoring qCl differences up to 0.003.

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Rule 1 applies for 14 out of 15 cases corresponding to a success rate of 93%,

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and thus appears to represent a powerful means for discriminating between active

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and non-active chloroaniline substrates with strain CBDB1. Taking 2-chloroaniline

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(A1 in Figure 4) as example, qCl is –0.046 and thus (with the 0.003 range of assumed

315

uncertainty) considered to be below –0.042, qualifying the Cl atom as resistant

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against CBDB1-mediated dechlorination. For C3 (3,5-DCA), the Hirshfeld charge is

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–0.052 for both (symmetrical) Cl substituents and thus even more below the thresh-

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old of –0.042 that would trigger abstraction by strain CBDB1. By contrast, E3 (PeCA)

319

features qCl values of –0.021 and –0.022, respectively, indicating that this congener

320

is active as CBDB1 substrate. The only counter example is D3 (2,4,6-TrCA) with qCl

321

= –0.038 and thus clearly above the threshold, but nevertheless dehalogenated only

322

by strain 14DCB1 (and not by strain CBDB1).

323

Rule 2 reflects a minority trend rather than a majority pattern. As can be seen

324

from Figure 4, B1, D1 and E2 making up 3/7 = 43% follow this regioselectivity rule.

325

By contrast, for the remaining four CBDB1-active substrates (C2, D2, E1, E3) a

326

systematic pattern could not be identified, although D2, E1 and E3 include metabo-

327

lites with the least negative Cl atom being removed. For D1 (2,3,4-TrCA) as example,


Page 15 of 31


15

328

the Cl atoms with qCl values of –0.030 and –0.033 are abstracted, whereas the Cl

329

atom attached to C4 with qCl = –0.050 resists strain CBDB1, but is removed by

330

14DCB1. The most pronounced counter example is C2 (3,4-DCA), where only the Cl

331

with the lower qCl (–0.056 vs –0.045) is cleaved by CBDB1 (and in fact also by strain

332

14DCB1).

333

Both rules refer to the electronic charge associated with the Cl substituents,

334

and thus suggest an initial attack of the CBDB1-dehalogenating enzyme at Cl, which

335

is discussed in more detail in the next section.

336

Hirshfeld charge vs 14DCB1-Mediated Dechlorination. As outlined above,

337

Dehalobacter sp. strain 14DCB1 yields a metabolite pattern significantly different

338

from the one obtained with Dehalococcoides mccartyi strain CBDB1. In Figure 4, the

339

Cl substituents experimentally active with 14DCB1 are marked through green or gold

340

color, with the latter being also cleaved by CBDB1. Comparative analysis of these

341

experimental results with the Hirshfeld charges obtained for H, Cl and chlorinated

342

carbon yield the following two rules:

343 344

Strain 14DCB1 Dechlorination Rule 1:

345

The chloroaniline is active as a strain 14DCB1 dehalogenation substrate if there is at

346

least one carbon-attached H with qH ≥ 0.047, ignoring qH differences up to 0.003.

347 348

Strain 14DCB1 Dechlorination Rule 2:

349

Regarding strain 14DCB1 regioselectivity, abstraction occurs preferably for Cl ortho

350

to the most positive H attached to the highest-charge (most positive or least nega-

351

tive) carbon, ignoring qCl, qH and qC differences up to 0.003.



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16

352 353

Examples for rule 1 are the 14DCB1-active substrates B1 (maximum qH of

354

0.044 equivalent within 0.003 uncertainty to the qH threshold of 0.047), and B2 (one

355

carbon-attached H with qH = 0.048), and A1 as chloroaniline resistant to 14DCB1

356

(maximum qH = 0.043). Here, the only counter example is D3 (2,4,6-TrCA) that is not

357

metabolized by 14DCB1 despite two H atoms with qH = 0.050. Accordingly, the over-

358

all success rate of this rule is 14/15 = 93%.

359

B1 (2,3-DCA) is a simple example for rule 2, because only one of the two Cl

360

atoms has an ortho H atom. In B2 (2,4-DCA), the most positive H atom is ortho to

361

both Cl atoms, but the one actually abstracted is attached to the carbon with the

362

higher Hirshfeld charge (qC 0.010 vs 0.005). Among the four Cl substituents of E2

363

(2,3,5,6-TeCA), only two are ortho to carbon-attached H and thus abstracted through

364

14DCB1, contrasting with the removal of the other two Cl substituents (red color in

365

Figure 4) upon exposure to CBDB1. This rule applies in 10 of 11 cases (all 14DCB1-

366

active substrates except D3 where the Cl attached to the most positive carbon resists

367

abstraction), corresponding to a success rate of 91%.

368

Interestingly, both rules involve the net atomic charge of the H atoms. In addi-

369

tion, the presence of at least one carbon-attached H atom has turned out as prere-

370

quisite for a possible 14DCB1 dechlorination activity of chloroanilines (see above).

371

These findings suggest that the 14DCB1 enzyme catalyzing the Cl abstraction may

372

proceed through initially attacking an H atom of the aromatic substrate, which con-

373

trasts with the presumed dehalogenation mechanism discussed so far in the litera-

374

ture35 as well as with the Cl charge as trigger of the dechlorination activity presently

375

observed with CBDB1.

376

For the presently analyzed chloroanilines, the Hirshfeld charges show only mi-


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17

377

nor variations across different functionals, basis sets and when including simulated

378

aqueous solvation (Figures S5-S7), confirming their relatively low degree of depen-

379

dence on the particular choice of the quantum chemical method.

380 381

382

DISCUSSION

383

Dechlorination Substrates. This study demonstrates that chloroanilines are

384

reductively transformed by Dehalococcoides mccartyi strain CBDB1 and Dehalobac-

385

ter sp. strain 14DCB1. However, the associated dehalogenation pathways differ, re-

386

sulting in different overall dehalogenation patterns. Chloroaniline congeners with

387

neighboring chlorines were transformed by Dehalococcoides mccartyi strain CBDB1,

388

while isolated chlorines were not removed (Figure 1, Figure S1 and Table 1). This

389

dechlorination pattern is in agreement with the ones of strain CBDB1 observed pre-

390

viously with chlorobenzenes,24, 36 and polychlorinated biphenyls37. The dehalogena-

391

tion of most chlorophenols26 and polychlorinated dibenzo-p-dioxins38 by this strain al-

392

so follow this pattern. Especially the case of the chlorinated dioxins emphasizes the

393

importance of the regioselectivity of dehalogenation: strain CBDB1 mainly removed

394

from 1,2,3,7,8-pentachlorodibenzo-p-dioxin the peripheral chlorine substituent form-

395

ing the most toxic dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin.38 In the pre-

396

sent study we confirm the finding of an earlier study16 with three chloroanilines that

397

the amino group in chloroanilines facilitates the dehalogenation of neighboring Cl

398

substituents in a way similar to the supporting function of the oxygen atoms in dioxin.

399

In addition, we certify that Cl substituents provide stronger support for dehalogena-

400

tion than amino substituents with strain CBDB1.

401

By contrast, Dehalobacter sp. strain 14DCB1 removed chlorine substituents ACS Paragon Plus Environment


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18

402

that are flanked at least by one hydrogen, i.e. doubly flanked chlorine substituents

403

were not removed. Translated to dioxins, this could prevent the formation of highly to-

404

xic intermediates, raising biotechnological interest in this dehalogenation pattern.

405

This dechlorination pattern was observed previously for the transformation of six dif-

406

ferent chlorobenzenes by strain 14DCB1.19 However, pentachlorobenzene, 1,2,3,5-

407

tetrachlorobenzene, 1,3,5-trichlorobenzene and 1,3-dichlorobenzene with hydrogen

408

in the aromatic ring were not metabolized by Dehalobacter sp. strain 14DCB1.19 The

409

persistence of these three chlorobenzenes against microbial transformation by strain

410

14DCB1 may be induced by the spatial structure of the relevant dehalogenase or

411

dehalogenases, which we have not clarified yet.

412

Reductive Dehalogenases. Both Dehalococcoides mccartyi strain CBDB1

413

and Dehalobacter sp. strain 14DCB1 are organohalide respiring microbes. Strain

414

CBDB1 cannot grow and transform organohalides without exogenous cobalamin.39

415

Similarly, Dehalobacter sp. E1 was not able to grow and dechlorinate β-hexachloro-

416

cyclohexane without the cobalamin offered by Sedimentibacter,22 suggesting that co-

417

balamin is an essential cofactor in the reductive dehalogenase of Dehalobacter spp.,

418

similar to other dehalogenating microorganisms.40-44 In addition, recent reports on the

419

structure of two dehalogenases provided direct evidence for the position of corrin co-

420

factors in reductive dehalogenases, suggesting electron transfer from cob(I)alamin to

421

organohalides as mechanism initiating the dehalogenation.35, 41 However, the struc-

422

ture of the dehalogenases from Dehalococcoides mccartyi strain CBDB1 and Deha-

423

lobacter sp. strain 14DCB1 have not been elucidated yet, which complicates the

424

comparison of mechanisms driving different dehalogenation pathways and patterns.

425

Alternatively, the electronic structure of terminal electron acceptors together with the

426

experimental determination of dehalogenation pathways can give insights into the

427

mechanism of dehalogenation. ACS Paragon Plus Environment

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19

428

Strain CBDB1 Substrates and Mechanism. Focusing on the substrates, the

429

quantum chemically calculated Hirshfeld31 net atomic charges provide a rationale for

430

the observed dehalogenation pathways of the 15 chloroanilines tested experimentally.

431

Corresponding solution-phase results yield a similar charge pattern (Figure S7),

432

keeping in mind that catalytic sites in protein pockets are usually not exposed to

433

aqueous solvation. Regarding strain CBDB1, the least negative Cl Hirshfeld charge

434

qCl discriminates active from non-active substrates in 14 out of 15 cases (93%). How-

435

ever, only for a subset of three active substrates the observed regioselectivity favors

436

the least negative Cl as primary site of attack (43%), although for some other sub-

437

strates removal of that Cl is included in one of several metabolic routes (see Figure

438

4). Moreover, the Cl net atomic charges are the only ones significantly related to the

439

CBDB1-mediated dehalogenation, confirming previous findings regarding homo- and

440

heterocyclic aromatics as CBDB1 substrates.16, 45 Accordingly and in line with a re-

441

cent study16 we hypothesize that for this bacterial strain, initial electron transfer from

442

cob(I)alamin to Cl initiates reductive abstraction of the latter, contrasting with a long-

443

held view about the aromatic carbon as primary site of enzyme attack.46

444

The present hypothesis is supported by the recently determined structure of

445

the heterologously expressed dehalogenase from Nitratireductor pacificus.35 Regard-

446

ing the electron transfer, at least two mechanistic variants would be compatible with

447

the experimental findings available so far: First, single electron transfer from cob(I)al-

448

amin to Cl could induce its abstraction as Cl–, and the aromatic radical intermediate

449

may receive a further electron from the intermediate cob(II)alamin and a proton to

450

form the reduced product (A in Scheme 1).16 Second, transfer to Cl of two electrons

451

from the supernucleophile cob(I)alamin leads to Cl– cleavage, and the resultant aro-

452

matic anion becomes protonated to yield the dechlorinated metabolite (B in Scheme

453

1). ACS Paragon Plus Environment


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20

454

Strain 14DCB1 Substrates and Mechanism. Various dechlorinated metabo-

455

lites generated by strain 14DCB1 differ from the ones obtained through strain CBDB1.

456

This can be traced back to the finding that with 14DCB1, the dehalogenation activity

457

appears to be triggered by the most positive carbon-attached H atom. This holds for

458

14 out of 15 cases (93%), and implies the presence of at least one unsubstituted aro-

459

matic carbon as necessary condition for the substrate activity. Moreover, the Hirsh-

460

feld charges can even predict the regioselectivity in 10 of 11 cases (91%), now in-

461

volving both the H atoms and the aromatic carbon bonded to the Cl that is being re-

462

moved. Again, corresponding solution-phase results as well as respective NPA char-

463

ges are available in Figure S3.

464

So far, the H atoms of unsubstituted aromatic carbons did not play a role in

465

hypotheses about the mechanism underlying the reductive dehalogenation of aroma-

466

tic halides. By contrast, literature considered the aromatic carbon of the substrate as

467

initial site of attack,46 which would make it difficult to rationalize the dechlorination re-

468

sistance of pentachloroaniline. The demonstrated impact of the presence and elec-

469

tronic charge of carbon-attached hydrogen on the activity and regioselectivity of de-

470

chlorination through strain 14DCB1, however, suggests the dehalogenase enzyme to

471

proceed through a primary attack at respective H atoms.

472

Associated possible one- and two-electron transfer mechanisms are outlined

473

in the lower half of Scheme 1. Transfer of one electron from Co(I) of cob(I)alamin to

474

the positively charged H atom could be transmitted along the H–CarCar–Cl unit to

475

cleave Cl–, and the intermediately formed aromatic radical may receive a further elec-

476

tron and a proton to form the dechlorinated product (C in Scheme 1). Alternatively, a

477

two-electron transfer to Cl with cleavage of Cl– could form an aromatic anion that be-

478

comes protonated to yield the reduced metabolite (D in Scheme 1).

479

In any case, the present findings suggest the carbon-attached H atom as priACS Paragon Plus Environment

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21

480

mary site of attack, which is compatible with previous reports about favorable inter-

481

actions between transition metals and hydrogen of aromatic rings.47,

482

viewpoint, supernucleophilic Co(I) could initially form an H-bond to a carbon-attached

483

H-atom, and then proceed with a full transfer of one or two electrons as outlined in

484

Scheme 1 (C and D).

48

From this

485

The two complementary modes of reductive dehalogenation catalyzed by De-

486

halococcoides mccartyi strain CBDB1 and Dehalobacter sp. strain 14DCB1, respect-

487

ively, provide opportunity to develop targeted strategies for biotechnological conver-

488

sion and biological remediation. Regarding a molecular-level understanding of the

489

underlying mechanisms, computational chemistry research into reaction intermedi-

490

ates and pathways may provide further insight as was demonstrated for the case of

491

cytochrome P450 catalysis.49-51

492 493

ASSOCIATED CONTENT

494

Supporting information

495 496

The Supporting Information is available free of charge on ACS Publications website at DOIW. Additional information as noticed in the text (PDF).

497 498

499

Notes The authors declare no competing financial interest.

500 501

ACKNOWLEDGEMENTS

502

L.A. is supported by the Deutsche Forschungsgemeinschaft in the frame of

503

the Forschergruppe FOR1530. Shangwei Zhang thanks Prof. Dr. Frank-Dieter KopinACS Paragon Plus Environment


Page 22 of 31

22

504

ke from the UFZ Department of Environmental Engineering for helpful discussions.

505

We thank Benjamin Scheer from the UFZ Department of Isotope Biogeochemistry for

506

technical chemical analyses.

507 508


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(37) Adrian, L.; Dudková, V.; Demnerová, K.; Bedard, D. L. “Dehalococcoides” sp. strain CBDB1 extensively dechlorinates the commercial polychlorinated biphenyl mixture Aroclor 1260. Appl. Environ. Microbiol. 2009, 75 (13), 4516-4524. (38) Bunge, M.; Adrian, L.; Kraus, A.; Opel, M.; Lorenz, W. G.; Andreesen, J. R.; Görisch, H.; Lechner, U. Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 2003, 421 (6921), 357-360. (39) Schipp, C. J.; Marco-Urrea, E.; Kublik, A.; Seifert, J.; Adrian, L. Organic cofactors in the metabolism of Dehalococcoides mccartyi strains. Phil. Trans. R. Soc. B 2013, 368 (1616), 20120321. (40) Parthasarathy, A.; Stich, T. A.; Lohner, S. T.; Lesnefsky, A.; Britt, R. D.; Spormann, A. M. Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS. J. Am. Chem. Soc. 2015, 137 (10), 3525-3532. (41) Bommer, M.; Kunze, C.; Fesseler, J.; Schubert, T.; Diekert, G.; Dobbek, H. Structural basis for organohalide respiration. Science 2014, 346 (6208), 455-458. (42) Kräutler, B.; Fieber, W.; Ostermann, S.; Fasching, M.; Ongania, K. H.; Gruber, K.; Kratky, C.; Mikl, C.; Siebert, A.; Diekert, G. The Cofactor of Tetrachloroethene Reductive Dehalogenase of Dehalospirillum multivorans Is Norpseudo‐B12, a New Type of a Natural Corrinoid. Helv. Chim. Acta 2003, 86 (11), 3698-3716. (43) Krasotkina, J.; Walters, T.; Maruya, K. A.; Ragsdale, S. W. Characterization of the B12- and Iron-Sulfur-containing Reductive Dehalogenase from Desulfitobacterium chlororespirans. J. Biol. Chem. 2001, 276 (44), 40991-40997. (44) Maillard, J.; Schumacher, W.; Vazquez, F.; Regeard, C.; Hagen, W. R.; Holliger, C. Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus. Appl. Environ. Microbiol. 2003, 69 (8), 4628-4638. (45) Lu, G.-N.; Tao, X.-Q.; Huang, W.; Dang, Z.; Li, Z.; Liu, C.-Q. Dechlorination pathways of diverse chlorinated aromatic pollutants conducted by Dehalococcoides sp. strain CBDB1. Sci. Total Environ. 2010, 408 (12), 2549-2554. (46) Banerjee, R.; Ragsdale, S. W. The Many faces of Vitamin B12: Catalysis by Cobalamin-Dependent Enzymes. Annu. Rev. Biochem. 2003, 72, 209-247. (47) Ribas, X.; Calle, C.; Poater, A.; Casitas, A.; Gómez, L.; Xifra, R. l.; Parella, T.; BenetBuchholz, J.; Schweiger, A.; Mitrikas, G. Facile C− H Bond Cleavage via a Proton-Coupled Electron Transfer Involving a C−HCuII Interaction. J. Am. Chem. Soc. 2010, 132 (35), 12299-12306. (48) Begum, R. A.; Day, V. W.; Kumar, M.; Gonzalez, J.; Jackson, T. A.; Bowman-James, K. M⋯H–C interaction – Agostic or not: A comparison of phenyl- versus pyridyl-bridged transition metal dimers. Inorg. Chim. Acta 2014, 417, 287-293. (49) Ji, L.; Schüürmann, G. Computational evidence for α-nitrosamino radical as initial metabolite for both the P450 dealkylation and denitrosation of carcinogenic nitrosamines. J. Phys. Chem. B 2012, 116 (2), 903-912. (50) Ji, L.; Schüürmann, G. Model and Mechanism: N‐Hydroxylation of Primary Aromatic Amines by Cytochrome P450. Angew. Chem. Int. Ed. 2013, 52 (2), 744-748; Angew. Chem. 125 (2), 772-776. (51) Ji, L.; Schüürmann, G. Computational biotransformation profile of paracetamol catalyzed by cytochrome P450. Chem. Res. Toxicol. 2015, 28 (4), 585-596.

663 664



Page 26 of 31

26

665

Tables

666 667

Table 1. Transformation of chloroanilines by Dehalococcoides mccartyi strain CBDB1 and

668

Dehalobacter sp. 14DCB1 measured with a whole-cell activity test. The cells of strain CBDB1

669

and 14DCB1 were grown in a medium with hexachlorobenzene and 1,2,3-trichlorobenzene,

670

respectively.a Dehalococcoides mccartyi strain CBDB1 Electron acceptor used

Products and their relative amount (%)

Specific enzymatic activity

(nkat/mg protein)

Dehalobacter strain. 14DCB1 Products and their relative amount (%)

Specific enzymatic activity

(nkat/mg protein)

2-MCA

nd

0

nd

0

3-MCA

nd

0

nd

0

4-MCA

nd

0

nd

0

2,3-DCA

3-MCA

2,4-DCA

6.7 ± 1.0

2-MCA

0.12 ± 0.01

nd

0

2-MCA

0.71 ± 0.02

2,5-DCA

nd

0

3-MCA

2.2 ± 0.1

2,6-DCA

nd

0

2-MCA

0.65 ± 0.03

3,4-DCA

3-MCA

3.7 ± 0.6

3-MCA

1.4 ± 0.2

3,5-DCA

nd

0

3-MCA

2.4 ± 0.2

2,3,4-TrCA

2,4-DCA (88.9),

37.9 ± 3.7

2,3-DCA

2.7 ± 0.2

24.6 ± 1.1

3,4-DCA (73.2) 2,5-DCA (26.8)

6.9 ± 0.5

3,4-DCA (11.1) 2,4,5-TrCA

2,5-DCA

2,4,6-TrCA

nd

3,4,5-TrCA

3,5-DCA (95.5),

0

2,4-DCA

0.48 ± 0.05

39.2 ± 7.4

3,4-DCA

0.29 ± 0.04

3,4-DCA (4.5) 2,3,5,6-TeCA

2,3,5-TrCA

nq

2,3,6-TrCA 2,3-DCA, 2,6-DCA

nq

PeCA

2,3,4,6-TeCA 2,4,6-TrCA

nq

nd

0

671

a

7

7

672

cells/mL for bacterial strain 14DCB1. A value of 30 fg protein per cell as described previously

673

late the protein amount from counted cell numbers. Abbreviations: nd, no product detected; nq, not quantified be-

674

cause no neat standard was available.

The cell density of the culture was about 1.9×10 cells/mL for bacterial strain CBDB1, and about 1.2×10 27


was used to calcu-

Page 27 of 31


27

675

Figures

676 677

Figure 1. Formation of dehalogenation products from diverse chlorinated anilines in cultures

678

of Dehalococcoides mccartyi strain CBDB1. Used terminal electron acceptors: (A) 2,3-DCA,

679

(B) 3,4-DCA, (C) 2,3,4-TrCA, (D) 2,4,5-TrCA, (E) 3,4,5-TrCA, (F) PeCA. All cultures initially

680

contained 40 µM, and were re-fed with 40 µM of the respective electron acceptor every

681

seven days.

682 683



Page 28 of 31

28

684 685

Figure 2. Formation of dehalogenation products from chlorinated anilines in cultures of

686

Dehalobacter sp. strain 14DCB1. Used electron acceptors: (A) 2,3-DCA, (B) 3,4-DCA, (C)

687

2,3,4-TrCA, (D) 2,4,5-TrCA, (E) 3,4,5-TrCA, (F) 2,3,5,6-TeCA. Inlays are magnifications of

688

the curves in cultures with low activity. Cultures initially contained 40 µM and were re-fed

689

with 40 µM of the respective electron acceptor every seven days. No products were found in

690

cultures with PeCA. The concentration of 2,3,6-TrCA in panel F was quantified using 2,4,5-

691

TrCA as standard.

692


Page 29 of 31


29

693 694

Figure 3. Dehalogenation pathways of chloroanilines in cultures with Dehalococcoides

695

mccartyi strain CBDB1 (A, green arrows) and Dehalobacter sp. strain 14DCB1 (B, blue ar-

696

rows), respectively. Whereas CBDB1 preferentially abstracts doubly-flanked Cl, 14DCB1 re-

697

moves Cl ortho to a carbon-attached H. Arrow coding: Bold = main pathway, ordinary = side

698

pathway, dashed = pathway to GC-unresolved products. Chemical structures in parentheses

699

are hypothesized as intermediates, but not commercially available for experimental confirma-

700

tion.

701



Page 30 of 31

30

702 703

Figure 4. Chloroaniline test compounds with Hirshfeld net atomic charges (B3LYP/Def2SVP)

704

and color-coded Cl atoms indicating abstraction only by Dehalococcoides mccartyi strain

705

CBDB1 (red), only by Dehalobacter sp. strain 14DCB1 (green), and by both CBDB1 and

706

14DCB1 (gold). Annotation “major” indicates the major site of dechlorination as observed

707

experimentally. The rows and columns are labeled by capital letters (A-E) and numbers (1, 2,

708

3), respectively, to facilitate compound identification (e.g. compound A2 is located in the top

709

middle position of the figure). ACS Paragon Plus Environment

Page 31 of 31


31

710

Schemes

711

712

713

Scheme 1. One- and two electron transfer mechanisms for the reductive dehalogenation of

714

chloroanilines catalyzed by Dehalococcoides mccartyi strain CBDB1 (A, B) and Dehalobacter

715

sp. strain 14DCB1 (C, D), respectively, employing 2,3-DCA as example substrate. The pre-

716

sumed electron donor is the supernucleophile Co(I) from cob(I)alamin.

717 718


Anaerobic Dehalogenation of Chloroanilines by Dehalococcoides

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