Data_Sheet_1_The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular.XLSX

<p>Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.</p

Interaction Mode and Regioselectivity in Vitamin B₁₂-Dependent Dehalogenation of Aryl Halides by <i>Dehalococcoides mccartyi</i> Strain CBDB1

The bacterium <i>Dehalococcoides</i>, strain CBDB1, transforms aromatic halides through reductive dehalogenation. So far, however, the structures of its vitamin B₁₂-containing dehalogenases are unknown, hampering clarification of the catalytic mechanism and substrate specificity as basis for targeted remediation strategies. This study employs a quantum chemical donor–acceptor approach for the Co­(I)-substrate electron transfer. Computational characterization of the substrate electron affinity at carbon–halogen bonds enables discriminating aromatic halides ready for dehalogenation by strain CBDB1 (active substrates) from nondehalogenated (inactive) counterparts with 92% accuracy, covering 86 of 93 bromobenzenes, chlorobenzenes, chlorophenols, chloroanilines, polychlorinated biphenyls, and dibenzo<i>-p-</i>dioxins. Moreover, experimental regioselectivity is predicted with 78% accuracy by a site-specific parameter encoding the overlap potential between the Co­(I) HOMO (highest occupied molecular orbital) and the lowest-energy unoccupied sigma-symmetry substrate MO (σ*), and the observed dehalogenation pathways are rationalized with a success rate of 81%. Molecular orbital analysis reveals that the most reactive unoccupied sigma-symmetry orbital of carbon-attached halogen X (σ_C–X^*) mediates its reductive cleavage. The discussion includes predictions for untested substrates, thus providing opportunities for targeted experimental investigations. Overall, the presently introduced orbital interaction model supports the view that with bacterial strain CBDB1, an inner-sphere electron transfer from the supernucleophile B₁₂ Co­(I) to the halogen substituent of the aromatic halide is likely to represent the rate-determining step of the reductive dehalogenation

Image_1_The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular.JPEG

<p>Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.</p

Anaerobic Ammonium Oxidation (Anammox) with Planktonic Cells in a Redox-Stable Semicontinuous Stirred-Tank Reactor

Anaerobic ammonium oxidizing (anammox) bacteria are routinely cultivated in mixed culture in biomass-retaining bioreactors or as planktonic cells in membrane bioreactors. Here, we demonstrate that anammox bacteria can also be cultivated as planktonic cells in a semicontinuous stirred-tank reactor (semi-CSTR) with a specific growth rate μ of 0.33 d^–1 at 30 °C. Redox potential inside the reactor stabilized at around 10 mV (±15 mV; vs standard hydrogen electrode) without gas purging. Reactor headspace pressure was used as a sensitive and real-time indicator for nitrogen evolution and anammox activity. The reactor was dominated by an organism closely related to “<i>Candidatus</i> Kuenenia stuttgartiensis” (∼87% abundance) as shown by Illumina amplicon sequencing and fluorescence <i>in situ</i> hybridization. Epifluorescence microscopy demonstrated that all cells were in their planktonic form. Mass balance analysis revealed a nitrite/ammonium ratio of 1.270, a nitrate/ammonium ratio of 0.238, and a biomass yield of 1.97 g volatile suspended solids per mole of consumed ammonium. Batch experiments with the reactor effluent showed that anammox activities were sensitive to sulfide (IC₅₀ = 5 μM) and chloramphenicol (IC₅₀ = 19 mg L^–1), much lower than reported for granular anammox biomass. This study shows that semi-CSTR is a powerful tool to study anammox bacteria

Reductive Dehalogenation of Oligocyclic Phenolic Bromoaromatics by <i>Dehalococcoides mccartyi</i> Strain CBDB1

Dehalococcoides mccartyi strains transform many halogenated compounds and are used for bioremediation. Such anaerobic transformations were intensively studied with chlorinated and simply structured compounds such as chlorinated benzenes, ethenes, and ethanes. However, many halogenated oligocyclic aromatic compounds occur in nature as either naturally produced materials or as part of commercial products such as pharmaceuticals, pesticides, or flame retardants. Here, we demonstrate that the D. mccartyi strain CBDB1 reductively debrominated two oligocyclic aromatic phenolic compounds, tetrabromobisphenol A (TBBPA) and bromophenol blue (BPB). The strain CBDB1 completely converted TBBPA to bisphenol A and BPB to phenol red with a stepwise removal of all bromide substituents. Debromination (but no cell growth) was detected in the cultures cultivated with TBBPA. In contrast, strain CBDB1 grew when interacting with BPB, demonstrating that this substrate was used as an electron acceptor for organobromine respiration. High doses of BPB delayed debromination and inhibited growth in the early cultivation phase. A higher toxicity of TBBPA compared with that of BPB might be due to the higher lipophilicity of TBBPA. Mass spectrometric analyses of whole-cell extracts demonstrated that two proteins encoded by the reductive dehalogenase homologous genes CbdbA1092 and CbdbA1503 were specifically induced by the used oligocyclic compounds, whereas others (e.g., CbdbA84 (CbrA)) were downregulated

Relative Contributions of <i>Dehalobacter</i> and Zerovalent Iron in the Degradation of Chlorinated Methanes

The role of bacteria and zerovalent iron (Fe⁰) in the degradation of chlorinated solvents in subsurface environments is of interest to researchers and remediation practitioners alike. Fe⁰ used in reactive iron barriers for groundwater remediation positively interacted with enrichment cultures containing <i>Dehalobacter</i> strains in the transformation of halogenated methanes. Chloroform transformation and dichloromethane formation was up to 8-fold faster and 14 times higher, respectively, when a <i>Dehalobacter</i>-containing enrichment culture was combined with Fe⁰ compared with Fe⁰ alone. The dichloromethane-fermenting culture transformed dichloromethane up to three times faster with Fe⁰ compared to without. Compound-specific isotope analysis was employed to compare abiotic and biotic chloroform and dichloromethane degradation. The isotope enrichment factor for the abiotic chloroform/Fe⁰ reaction was large at −29.4 ± 2.1‰, while that for chloroform respiration by <i>Dehalobacter</i> was minimal at −4.3 ± 0.45‰. The combined abiotic/biotic dechlorination was −8.3 ± 0.7‰, confirming the predominance of biotic dechlorination. The enrichment factor for dichloromethane fermentation was −15.5 ± 1.5‰; however, in the presence of Fe⁰ the factor increased to −23.5 ± 2.1‰, suggesting multiple mechanisms were contributing to dichloromethane degradation. Together the results show that chlorinated methane-metabolizing organisms introduced into reactive iron barriers can have a significant impact on trichloromethane and dichloromethane degradation and that compound-specific isotope analysis can be employed to distinguish between the biotic and abiotic reactions involved

Anaerobic Microbial Transformation of Halogenated Aromatics and Fate Prediction Using Electron Density Modeling

Halogenated homo- and heterocyclic aromatics including disinfectants, pesticides and pharmaceuticals raise concern as persistent and toxic contaminants with often unknown fate. Remediation strategies and natural attenuation in anaerobic environments often build on microbial reductive dehalogenation. Here we describe the transformation of halogenated anilines, benzonitriles, phenols, methoxylated, or hydroxylated benzoic acids, pyridines, thiophenes, furoic acids, and benzenes by <i>Dehalococcoides mccartyi</i> strain CBDB1 and environmental fate modeling of the dehalogenation pathways. The compounds were chosen based on structural considerations to investigate the influence of functional groups present in a multitude of commercially used halogenated aromatics. Experimentally obtained growth yields were 0.1 to 5 × 10¹⁴ cells mol^–1 of halogen released (corresponding to 0.3–15.3 g protein mol^–1 halogen), and specific enzyme activities ranged from 4.5 to 87.4 nkat mg^–1 protein. Chlorinated electron-poor pyridines were not dechlorinated in contrast to electron-rich thiophenes. Three different partial charge models demonstrated that the regioselective removal of halogens is governed by the least negative partial charge of the halogen. Microbial reaction pathways combined with computational chemistry and pertinent literature findings on Co^I chemistry suggest that halide expulsion during reductive dehalogenation is initiated through single electron transfer from B₁₂Co^I to the apical halogen site

Phylogenetic tree of <i>Dehalococcoidia</i> (DEH) 16S rRNA gene sequences highlighting the phylogenetic position of the 11 OTUs obtained from Lake Pavin water column.

<p>The phylogenetic analysis was performed using the neighbor-joining method; branches with bootstrap values (1000 replicates) of at least 50% are marked with a dot. The tree was constructed using MEGA version 6.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref038" target="_blank">38</a>]. The branches were coloured using iTOL tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref045" target="_blank">45</a>]. Sequences from this study fall into three classes defined by Wasmund <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref015" target="_blank">15</a>]: GIF-9B in dark blue, MSBL5 in light blue and Ord-DEH in orange (<i>Dehalogenimonas</i>) and in yellow (<i>Dehalococcoides</i>). An additional class not previously described is presented in grey as an annotated new subgroup. Sequences of cultured DEH are highlighted in red. Sequences derived from single-cell genome DEH-J10 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145558#pone.0145558.ref016" target="_blank">16</a>] and an aquifer sediment metagenome-derived genome RBG-2 are highlighted in blue. The scale bar represents 1% sequence divergence.</p

Quantification of ‘the total density of’ bacterial 16S rRNA genes and of two DEH-related phylotypes in Lake Pavin water column.

<p>Bacteria are represented by filled triangles. For DEH-related phylotypes: OTU1 is represented by filled diamonds and a dashed line and OTU2, by filled squares and a dotted line. Shown are mean values and standard deviations of triplicate PCR reactions.</p

Genomic fragments obtained using the specific-gene capture approach targeting an <i>rdhA</i> sequence identified in a Lake Pavin metagenome [39].

<p>The genomic fragments were recovered from the 68 m-depth sample by specific-gene capture approach using probes targeting a <i>rdhA</i> gene (first and second capture experiments) and a hypothetical protein (second capture experiment) identified in Lake Pavin water column. Capt_rdase, RdaseH8-1, RdaseH8-2, RdaseH8-3 and RdaseH8-4: capture probes.</p