This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. pubs.acs.org/jpr Article Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M Alba Trueba-Santiso,⊥ Kenneth Wasmund,⊥ Jesica M. Soder-Walz, Ernest Marco-Urrea,* and Lorenz Adrian Cite This: J. Proteome Res. 2021, 20, 613−623 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146−242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron−sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone- independent respiration in Dehalogenimonas. KEYWORDS: organohalide respiration, dihaloelimination, Dehalogenimonas alkenigignens strain BRE15M, Dehalococcoidia, genome sequencing, 1,2-dichloropropane, proteome profiling ■ INTRODUCTION Organohalides are common soil and groundwater pollutants. genes: rdhA, encoding for the catalytically active subunit RdhA, and rdhB encoding a putative membrane anchor RdhB.5 RdhA Although they can be naturally produced, the high proteins are usually located attached to the cytoplasmic concentrations found at many industrial sites are due to membrane facing the outer surface, indicating the need for improper storage or disposal practices and represent a threat to both, the TAT-leader peptide and the putative membrane- human and environmental health because of the toxicity or anchoring RdhB protein. carcinogenicity of many of them.1 A potential solution to treat In many OHRB, quinones play an important role as electron sites impacted with organohalides relies on organohalide- carrier between electron supplying protein complexes and the respiring bacteria (OHRB), which can use organohalides as RDase (e.g., in Sulfurospirillum or Dehalobacter).6 In these electron acceptors to harness energy and the reduction often cases, energy is conserved via the quinone-mediated transport results in decreased toxicity of the contaminants.2 of protons across the membrane.6 In contrast, the genomes of Reductive dehalogenation is catalyzed by enzymes termed the obligate OHRB Dehalococcoides spp. lack the genes for reductive dehalogenases (RDases). Respiratory RDases in complete biosynthesis of quinones.6−8 In quinone-independent OHRB have conserved features in their amino acid sequence reductive dehalogenation, energy conservation was suggested including a twin arginine translocation (TAT)-leader peptide that tags the protein for transport to and/or across the Received: July 23, 2020 cytoplasmic membrane, and two iron−sulfur cluster binding Published: September 25, 2020 motifs.3 Almost all characterized RDases contain a corrinoid in the active center, although corrinoid-binding motifs have seldom been found in the amino acid sequences.4 Typically, RDases are encoded in operons composed by at least two © 2020 American Chemical Society https://dx.doi.org/10.1021/acs.jproteome.0c00569 613 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research ■ pubs.acs.org/jpr Article to be coupled to the transfer of electrons from a hydrogenase MATERIALS AND METHODS to a complex iron−sulfur molybdoenzyme (CISM).9 Recent Cultivation of the Dehalogenimonas-Containing Culture studies have described a quinone-independent respiratory chain in Dehalococcoides mccartyi strain CBDB1, catalyzed by A Dehalogenimonas-containing sediment-free culture trans- forming 1,2-DCP to propene was maintained for more than an organohalide respiration complex (OHR complex) that 5 years in our laboratory as described elsewhere.20 Each directly transfers the electrons derived from H2 oxidation by microcosm contained 70 mL of an anaerobic bicarbonate- hydrogen uptake hydrogenase (Hup) via a CISM to the buffered medium, reduced with Na2S·9H2O and L-cysteine RDase.10 In this proposed scheme, Fe−S clusters in several of (0.2 mM each), amended with 5 mM sodium acetate as carbon the multiprotein complex subunits act as an electron relay source, and gassed with N2/CO2 (4:1, v/v, 0.2 bar over- between hydrogenase and RDase.9,11 According to Seidel et pressure) and H2 (added to an overpressure of 0.4 bar). All al.,11 the new nomenclature OmeA/OmeB was proposed for microcosms were cultivated under static conditions in the dark the CISM α- and γ-subunit, standing for organohalide at 25 °C. To enrich the Dehalogenimonas population, we respiration-involved molybdoenzyme. HupX, encoded in the followed a dilution-to-extinction procedure24 together with the hup operon in Dehalococcoides, interacted strongly with OmeA addition of streptomycin (50 mg L−1) and vancomycin (5 mg and OmeB, forming a tight module. A much less strong L−1). Cultures were transferred into a fresh medium with an interaction was found between the OmeA/OmeB/HupX inoculum of 10% (v/v) after the dechlorination of two to three module and a hydrogenase module HupS/HupL. doses (100−500 μM) of 1,2-DCP. Bacteria of the genus Dehalogenimonas are obligate OHRB For genome sequencing, 420 mL of this anaerobic enriched culture was used growing on 1,2-DCP as the only halogenated (class Dehalococcoidia), and were known for their potential to electron acceptor for 34 transfers (10% v/v). For proteomic use vicinally halogenated alkanes.12 Recently, isolated studies, cultures growing on 1,2-DCP for >42 transfers (10% Dehalogenimonas strains have been shown to also respire v/v) were used after the dechlorination of at least 2 mM 1,2- with chlorinated ethenes and benzenes.13,14 To date, the DCP. genomes of five Dehalogenimonas strains have been sequenced: Gas Chromatographic Analysis (i) Dehalogenimonas lykanthroporepellens strain BL-DC-9T,15 1,2-DCP and propene were analyzed from headspace samples (ii) Dehalogenimonas alkenigignens strain IP3-3,16 (iii) with a gas chromatograph (GC) model 6890N (Agilent Dehalogenimonas strain WBC-2,17 (iv) Dehalogenimonas Technologies; Santa Clara, USA) equipped with an HP-5 formicexedens strain NSZ-14T,18 and (v) “Candidatus column (30 m × 0.32 mm with 0.25 μm film thickness; Agilent Dehalogenimonas etheniformans” strain GP.13 Only three Technologies) and a flame ionization detector. Helium was proteins involved in organochlorine respiration have been used as the carrier gas (1.5 mL min−1). The injector and characterized in Dehalogenimonas spp.: (i) DcpA has been detector temperatures were both set at 250 °C. After the shown to dechlorinate 1,2-dichloropropane (1,2-DCP) to injection of the sample (split ratio = 2:1), the initial oven propene,19 (ii) TdrA was responsible for the hydrogenolysis of temperature (40 °C), ramped at 10 °C min−1 to 50 °C, and trans-dichloroethene to vinyl chloride,17 and more recently, then ramped at 20 °C min−1 to 120 °C. Both compounds were (iii) CerA was shown to catalyze the dechlorination of vinyl identified and quantified using external chemical standards. chloride to ethene.13 The sequenced Dehalogenimonas Nominal concentrations of 1,2-DCP and propene are ex- genomes harbor 22−52 rdhA genes, and in contrast to all pressed as μmol per L of liquid volume or in nanomoles per other known OHRB, most of them lack colocated genes for bottle or vial. membrane-anchoring protein cognates (rdhB), a feature that Metagenomic Sequencing and Genomic Analysis remains unexplained. Details regarding cell harvesting, DNA extraction, genome Previous work in our laboratory focused on enriching a sequencing, and assembling are provided in the Supporting nonmethanogenic Dehalogenimonas-containing culture from Information Materials and Methods section. A final automatic sediment samples collected from the Besòs River estuary (Sant annotation was performed using the MicroScope annotation Adrià de Besòs, Barcelona, Spain), which appeared to pipeline.25 Locus numbers used in this article are derived from exclusively transform vicinally halogenated alkanes via the MicroScope annotation. The locus numbers are prefixed dihaloelimination.20 The compounds transformed included with “BRE15M_v1_” and an accompanying number, whereby the first two digits denote the scaffold number, and the last 1,2-DCP, 1,2,3-trichloropropane, 1,2-dichloroethane, 1,1,2- four digits denote the gene number of each particular scaffold. trichloroethane, 1,1,2,2-tetrachloroethane, and 1,2-dibromo- Predicted proteins were searched for signal peptides mediating ethane.20−23 Here, we provide biochemical data on the the export from the cytoplasm using SignalP-5.0.26 Trans- molecular organization of the proteins involved in the membrane helices were detected using TMHMM server.27 respiratory chain of this novel Dehalogenimonas strain. For Subcellular location predictions were performed using this, we obtained a high-quality draft genome sequence of PSORTb server.28 The genome is available at GenBank strain BRE15M and applied a combination of proteome under accession QEFQ00000000. The genome and annota- profiling, blue native polyacrylamide gel electrophoresis tions are also publicly accessible in the MicroScope platform (BNE), dehalogenation activity assays and protein mass (https://mage.genoscope.cns.fr/microscope/home/index.php, spectrometry. In line with recent studies with D. mccartyi accession WGS BRE15M.1). strain CBDB1, our data suggest the existence of a quinone- Protein Profiling independent respiratory dehalogenase protein complex in For cell harvesting, triplicate 140 mL samples of a 1,2-DCP Dehalogenimonas, thereby extending the phylogenetic occur- dechlorinating Dehalogenimonas culture were separately cen- rence of these complexes outside of the genus Dehalococcoides. trifuged at 8000g and 10 °C to a final volume of 1 mL. A 614 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article detailed protocol is provided in the Supporting Information acetone stock solution with a glass syringe (Hamilton) to a Materials and Methods section. Cells were lyzed by freeze− final concentration of 200 μM 1,2-DCP. thawing (−20 °C/+20 °C, 6×) and sonication for 30 s. The For each activity measurement from BNE gels, different samples were then concentrated by ultrafiltration with Amicon controls were included. As a positive control, 25 μL of the Ultra 0.5 mL centrifugal filter units with a 10 kDa cutoff crude protein extract were added to the assay buffer instead of (Millipore) at 14,000g for 30 min at 10 °C. After a slice. Also, controls with either 25 μL supernatant of the centrifugation, filters were turned upside down and the protein solubilized protein extract or the remaining pellet after extract was spun off for 2 min at 1000g, 10 °C. Protein solubilization were included to evaluate the efficiency of the concentrations in the obtained samples were quantified by the membrane protein extraction. To control for abiotic trans- Bradford method. All triplicate samples were then processed by formation of 1,2-DCP, parallel negative controls containing the in-solution digestions for nano-liquid chromatography tandem assay buffer (i) without cells or gel slices from outside the area mass spectrometry (nLC−MS/MS) as described below. where proteins were loaded and (ii) with 25 μL of crude Blue Native Gel Electrophoresis extract but no 1,2-DCP was included to test the production of For each BNE gel, 140 mL samples of a 1,2-DCP propene from other sources than 1,2-DCP. All controls were dechlorinating Dehalogenimonas culture were centrifuged as performed in triplicate. described above. One milliliter of aliquoted concentrated cells The activity test vials were thoroughly mixed and incubated was centrifuged again at 9000g for 20 min and 800 μL of the upside down inside the glove box, at 30 °C without shaking. supernatant was removed. Then, 1× PBS buffer (pH 7.2) was After 24 h, the headspace of each sample was analyzed for added to a final volume of 600 μL. Cell density was quantified propene production by gas chromatography. at this point by direct epifluorescence microscopic cell Protein Identification counting on slides covered with agarose as previously Silver stained bands were cut from BNE gels, the bands were described.29 Then, cells were lysed by a bead beater (FastPrep FP120, Thermo) at speed 4 m s−1, in six cycles of 40 s beating washed with ddH2O and destained in a 1:1 (v/v) mixture of 30 alternated with 1 min resting in ice. Membrane proteins were mM K3[Fe(CN)6] and 100 mM Na2O3S2. Destained BNE solubilized by adding n-dodecyl β-D-maltoside (DDM) to a bands and in-solution samples were treated as described concentration of 0.05, 0.1, or 1% w/v, and incubating the previously to reduce and alkylate cysteine residues.11 Protein samples at 10 °C for 1 h with gentle shaking under anaerobic digestion from both in gel and in-solution samples was done conditions. After DDM incubation, samples were centrifuged overnight at 37 °C on a shaker after the addition of 0.1 μg of at 16,600g for 45 min at 4 °C. The supernatant (DDM- porcine trypsin (Proteomics Sequencing Grade, Promega). solubilized protein extract) was amended with 0.125% w/v The obtained peptides were extracted from the slices by 10 Coomassie G-250 additive and loaded in triplicate wells (25 min incubation in a solution containing formic acid 5% (v/v) μL/well) onto a precasted 4−16% gradient Bis-Tris gel and acetonitrile 50% (v/v) three times and afterward extracts (NativePAGE Novex, Invitrogen) and run inside an anaerobic were combined. Extracts from both in gel and in-solution glove box, maintaining the BNE system in contact with ice samples were dried, resolubilized in 0.1% of formic acid packs. Light blue cathode buffer and anode buffer were solution, and desalted using ZipTip-μC18 material (Merck prepared following the manufacturer’s instructions, degassed, Millipore). and cooled down to 4 °C before the electrophoresis was Peptides were analyzed by nLC−MS/MS on a nanoUPLC started. NativeMark Unstained Protein Standard (Invitrogen) system (nanoAcquity, Waters) hyphenated via a TriVersa was used as protein ladder. Electrophoresis was run at 150 V NanoMate (Advion, Ltd., Harlow, UK) to an Orbitrap Fusion for 60 min, and then at 200 V for 30 min more. Once the mass spectrometer (Thermo Scientific) as described pre- electrophoresis was finished, one of the replicate lanes and the viously.11 Glyceraldehyde-3-phosphate dehydrogenase from protein ladder lane were cut from the gel using a scalpel and Staphylococcus aureus was used as internal standard and was the protein bands were stained for visualization by a short added to the gel slices before derivatization. silver staining method.30 The rest of the gel was stored in Peptide identification was conducted by Proteome Discov- anode buffer at 4 °C. erer (v2.2, Thermo Fisher Scientific) using SequestHT as a Dehalogenase Activity Assays search engine and a FASTA-file of proteins from the draft To test RDase enzyme activity, specific assays were set-up genome of the Dehalogenimonas strain in the culture as a inside an anoxic glove box.31 Anoxic 10 mL glass vials were database. False discovery rates for peptide identification of 1% used, containing 2 mL of an assay buffer with 200 mM was set as threshold, calculated versus a decoy database using potassium acetate buffer (pH 5.8), 2 mM methyl viologen, and the percolator node. Label free quantification of the detected 2 mM titanium(III) citrate [2 mM in respect to titanium(III)] proteins was performed with the abundance value obtained and amended with 200 μM of 1,2-DCP. For the whole-cell from Proteome Discoverer Minora node. The relative protein activity assays, 200 μL of a cell concentrate (109 to 1010 cells abundance in this study was defined as the abundance of a mL−1) were inoculated to each assay vial. These experiments protein in one slice relative to the total abundance of this were performed in triplicates. protein across all slices of the BNE gel lane. To detect the To measure activity in BNE gels, a nonstained lane from the selenocysteine residue in OmeA different versions of the BNE was cut into slices using a steel scalpel. Each slice was protein with a selenocysteine, a cysteine, or a serine instead of introduced into a 2 mL assay vial. Slices were smashed into the TGA stop codon were added to the database and the smaller pieces to better expose proteins to the assay buffer. The search algorithm was adapted to detect selenocysteine as a vials were closed with Teflon-coated rubber septa and modification of cysteine. Results were confirmed by manually aluminum crimps and amended with 1,2-DCP from a 1 mM examining the respective MS2 spectra. 615 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article Figure 1. Phylogenetic tree showing the relationship of D. alkenigignens strain BRE15M to other strains of Dehalogenimonas and D. mccartyi strain 195 using a phylogenomic approach. Phylogenetic analysis was performed using an alignment of concatenated protein sequences of 34 single copy marker genes from CheckM analyses.32 The tree was constructed using default parameters using FastTree (version 2.1.10).33 The scale bar represents 5% sequence divergence. GenBank accession numbers are in parentheses. Table 1. List of RdhA Proteins Detected in Full Protein Extracts of D. alkenigignens Strain BRE15M Grown with 1,2-DCP as Only Halogenated Substrate for More Than 35 Transfersa replicate 1 replicate 2 replicate 3 accession abundance rank abundance rank abundance rank BRE15M_v1_160008 (DcpA) 7.34 × 107 88 2.40 × 108 10 3.44 × 108 7 BRE15M_vl_160007 4.55 × 107 134 7.95 × 107 41 1.32 × 108 30 BRE15M_v1_40117 3.46 × 107 162 1.19 × 107 375 BRE15M_v1_30017 2.17 × 107 246 1.27 × 107 276 1.77 × 107 300 BRE15M_v1_10074 5.44 × 106 435 1.32 × 107 348 BRE15M_v1_150026 6.46 × 105 808 2.51 × 106 583 2.99 × 106 654 BRE15M_v1_30093 1.11 × 106 724 BRE15M_v1_30016 2.48 × 106 622 5.84 × 105 823 1.34 × 105 1086 BRE15M_v1_30086 1.22 × 105 896 9.75 × 104 940 1.82 × 106 746 BRE15M_v1_170013 9.49 × 104 1100 protein concentration in the sample in mg/mL 6.9 2.14 5.08 number of detected proteins 901 947 1114 sum of abundances of all detected proteins 2.74 × 1010 1.84 × 1010 2.61 × 1010 a The rank columns show the rank of a protein relative to all proteins in that sample, calculated from the abundance values. Empty cells indicate that a protein was not detected in this replicate. ■ RESULTS Draft Genome Sequence of D. alkenigignens BRE15M following section the MicroScope gene locus tag “BRE15M_v1” will be omitted) were apparently truncated because of being at contig break. Only eight rdhA had cognate Metagenome sequencing was performed on DNA extracted genes for membrane-anchoring proteins encoded directly from the enrichment culture to investigate the genomic adjacent to the rdhA (Table S2). In total, six rdhB were properties of the Dehalogenimonas strain and to allow for automatically predicted, and we manually identified three further proteomic studies. A high-quality draft genome of a additional possible rdhB that were adjacent to rdhA and Dehalogenimonas sp. was obtained consisting of a total of encoded proteins with transmembrane helixes that may anchor 1,654,503 bp within 18 contigs, a GC content of 56.3%, and RdhA proteins into membranes (Table S3, Figure S1). containing 1795 predicted protein coding genes, all similar to Interestingly, one rdhB gene (_030121) was found at a locus reported Dehalogenimonas genomes (Table S1). CheckM where no rdhA was present, the closest being over 22 kbp results indicated that the genome was 97.8% complete with away. This brought into question whether the encoded protein no contamination (0%).32 In line with previous 16S ribosomal may be functional as an RdhB. We therefore searched for RNA (rRNA) gene sequencing of cultures from this enrich- closest homologs of this protein in other Dehalococcoidia. ment,20 the 16S rRNA gene of the Dehalogenimonas genome Although the closest homologs to this RdhB in other was 100% identical to the 16S rRNA gene of D. alkenigignens Dehalogenimonas spp. were also not adjacently encoded to an rdhA gene, several homologs with >80% amino acid identity in strain IP3-3, suggesting the strain in our culture belongs to the multiple D. mccartyi strains (CBDB1, DCMB5 and VS) species D. alkenigignens. Average nucleotide identity (gANI) of harbored an adjacently encoded RdhA. Together this suggests the whole genome to D. alkenigignens strain IP3-3 showed locus _030121 encodes a functional RdhB. 98.7% ANIb (BLASTN) identities from approximately 92% of The genome encoded various uptake and cytoplasmic the regions that aligned. We denominated our strain D. hydrogenases, formate dehydrogenases and a possible OHR alkenigignens strain BRE15M. The close similarity with D. complex, similar to previously described in Dehalococcoides alkenigignens was further supported by phylogenetic analysis of spp.9,11 No genes for biosynthesis of quinones or cytochromes concatenated protein sequences derived single copy marker were found. Further details regarding gene annotations and genes (Figure 1). Reciprocal BLASTP analyses of protein protein properties are presented in the Supporting Information sequences from D. alkenigignens strains BRE15M and IP3-3 Results section. suggested no major functional differences exist between the strains (Supporting Information Results section). Proteome Profiling The genome contains 31 full-length rdhA genes that were The shotgun proteomic analysis of strain BRE15M grown with not truncated by contig breaks, while two additional partial 1,2-DCP identified a total of 1152 proteins, which represented rdhA (BRE15M_v1_070029 and BRE15M_v1_150066; in the the 64% of the annotated proteins in strain BRE15M (Table 616 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article Figure 2. Distribution of dehalogenating activity in BNE with protein membrane samples of strain BRE15M solubilized with 0.05, 0.1 or 1% (w/v) DDM after lysis by bead beating. Dehalogenating activity was measured in nanomoles of propene produced from 1,2-DCP after 24 h of incubation. Data shows means of duplicate cultures ±SD. Dehalogenating activity in the corresponding controls is shown in Table S14. Figure 3. Distribution of dehalogenating activity in BNE with protein samples of strain BRE15M solubilized with 0.1% (w/v) DDM after lysis by bead beating. Dehalogenating activity was measured as nmol propene derived from 1,2-DCP dihaloelimination detected in enzyme assays within gel slices. Data shows means of duplicate culture samples ±SD. Molecular weight ranges were estimated from gel images using Image Lab software. “M”: protein molecular mass marker. S12). A total of 10 RDases were expressed in strain BRE15M OHR complex: OmeA (_040002/_040003), HupX when grown with 1,2-DCP (Tables 1 and S12). The most (_040001) and OmeB (_130001)9,11 were detected in each abundantly expressed RDase (_160008) was an ortholog of the of the protein extract triplicates of strain BRE15M with >106 RDase DcpA, which catalyzes reduction of 1,2-DCP to abundance. None of the few annotated genes involved in heme propene in D. mccartyi, Dehalogenimonas and uncultured or quinone biosynthesis were expressed, further supporting the bacteria. The RDase _160008 had >90% amino acid sequence conclusion that strain BRE15M is independent from identity to DcpA (Table S13).19 The second most abundant cytochromes and quinones. Finally, heterodisulfide reductase RDase (_160007) is encoded adjacently to the DcpA ortholog A and B subunits (_010277 and _010278) were expressed in and its putative membrane-anchoring protein DcpB strain BRE15M, proteins that are not encoded in D. mccartyi (_160009) (Figure S2). Interestingly, _160007 is one of the strains.13 four RDases which lacks the TAT signal peptide and has a Detection of the OHR Complex of Strain BRE15M by BNE predicted cytoplasmic location according to Phobius34 (Table When membrane proteins of strain BRE15M were solubilized S2). in 1% DDM without an additional cell disruption step and A membrane-bound [NiFe] hydrogenase (HupL/HupS, separated by BNE, dehalogenating activity was detected in gel _070001, _070002), was expressed at high abundance slices corresponding to a molecular mass range between 146 (absolute abundance > 106) as described for D. mccartyi and 242 kDa (Table S14). The activity produced low amounts strains.9,11,35 Also other proteins reported to be part of the of propene from 1,2-DCP by dichloroelimination. No defined 617 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article Figure 4. Relative abundance of proteins predicted to be associated with the OHR complex at different molecular weights in BNE gels after solubilization of strain BRE15M cells at different concentrations of DDM (panel A: 1% DDM, panel B: 0.1% DDM, panel C: 0.05% DDM). bands were observed in the gel after silver staining (not To narrow down the molecular range of the maximum shown), and most of the dehalogenating activity detected in dehalogenating activity, two replicate gel lanes with 0.1% the controls remained in the pellet control (390.14 ± 1.77 DDM were cut into slices of ∼2 mm across the mass range of nmol propene), compared to the protein extract supernatant 63−490 kDa and were subjected to activity assays (Figure 3). (146.93 ± 0.75 nmol propene). Together, this indicated Maximum activity was detected in slices VI and VII in both inefficient protein extraction (Table S14). gels, corresponding to molecular weights of 179−229 kDa. Next, cell disruption by bead beating combined with These results strongly support the presence of a organohalide solubilization with three different DDM concentrations was respiratory complex in strain BRE15M. tested (0.05, 0.1 and 1% DDM). The detailed conditions for Mass Spectrometric Analysis of BNE Slices these experiments are shown in Table S15. The resolution of the protein bands in the native gel was different among the Each of the six analyzed regions of the stained gels with 0.05, three conditions tested (Figure 2). With 0.05% DDM, the 0.1 and 1% DDM were subjected to in-gel trypsin digestion observed bands were very weak and blurry. On the contrary, in and subsequently analyzed by nLC−MS/MS. The number of 1.0% DDM bands were defined and intense, however, the unambiguously identified proteins for each gel region ranged bottom part of the gel (lower molecular weight) repeatedly from 175 to 966 (Figure S3). Thirteen RdhA paralogs were showed a blue Coomassie accumulation that impeded the detected in the gel experiment treated with 0.05% DDM, 10 visualization of the bands located below. With 0.1% DDM with 0.1% DDM and 9 with 1% DDM (Tables S16−S18). In bands were well defined along the whole lane. Parallel accordance with the production of propene detected in the in- unstained gel lanes with the three DDM concentrations were gel dehalogenating activity tests, the DcpA ortholog predicted cut into six slices each, following the stained molecular mass to be involved in 1,2-DCP dihaloelimination was abundantly ruler marks and each section was subjected to a dehalogenase detected in gel regions from the molecular range 146−242 kDa enzyme activity test with 1,2-DCP as electron acceptor. (Figure S4). Interestingly, the RDase with locus tag _160007 Propene was detected in all gel slices corresponding to a was the most abundant RDase expressed in the slices with molecular weight of 146−242 kDa in the three gel lanes. maximum dechlorinating activity in the gels with 0.1 and Activity was also detected in gel slices at 242−480 kDa in gels 0.05% (Figure S4). with 0.1 and 1% DDM, and at 66−146 kDa in the gel with In addition to RDases, we detected several other proteins in 0.1% DDM (Figure 2). Overall, a slight shift of the reductive the gel slices in which the OHR complex was detected (full dehalogenation activity toward higher molecular weight lists are given in Tables S16−S18). The DcpA ortholog and fractions was observed at higher DDM concentration. Notably, the RDase with the locus tag _160007 comigrated with the two maximum dehalogenating activity was detected in the hydrogenase subunits HupS and HupL, the OmeA, the molecular weight range between 146 and 242 kDa with 0.1% putative membrane-integrated anchor OmeB, and the four- DDM (Figure 2). Dehalogenating activity was never detected FeS cluster protein HupX, in gel regions that showed below 66 kDa in any of the experiments, indicating that maximum in-gel dehalogenating activity (regions 3−4 in monomeric RdhA protein (∼50 kDa) was part of a higher proteins solubilized with 1% DDM, and region 3 in proteins molecular mass respiratory complex or that it lost its activity as solubilized with 0.05 or 0.1% DDM) (Figures 2 and 4). These a monomeric enzyme. eight proteins showed a clear maximum of relative protein 618 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article Figure 5. Relative abundance of proteins associated with the OHR complex at different molecular weights in BNE gels after solubilization of strain BRE15M cells at 1% DDM (panel A), 0.1% DDM (panel B), and 0.05% DDM (panel C). abundance in region 3 in the gel with 0.1% DDM-extracted abundance in band VII (Figure 5A), which coincides with the proteins. OmeA/OmeB/HupX module. Potentially supporting this Only one RdhB protein was identified with a single peptide physical association, the hppA gene was encoded within the in 0.1 and 1% DDM BNE slices being colocalized with the same gene neighborhood as the omeA and hupX genes in DcpA ortholog (Figure 4A,B). This RdhB protein with the Dehalogenimonas strains BRE15M and IP3-3. locus tag _160009 is encoded in the same operon as DcpA in The same procedure was followed for dominant protein strain BRE15M (Figure S2) and shares 88% sequence identity bands in a silver-stained gel lane with 0.1% DDM-solubilized (e-value 3 × 10−40) with the DcpB (locus tag Dehly_1525) in proteins focusing on the region 66−480 kDa (Figure S5B). All D. lykanthroporepellens strain BL-DC-9, predicted to function of the proteins suspected to be part of the respiratory complex as a membrane anchor for DcpA. Interestingly, the topology of were detected in band VII (205−229 kDa) with the exception DcpB in strain BRE15M differs from the majority of the RdhB of DcpB, and in band VI (179−205 kDa) with the exception of reported so far, with two predicted transmembrane regions DcpB and HupS (Figure 5B, Table S20). These bands VI and instead of three (Figure S1). In summary, the identified VII corresponded to the unstained parallel gel slices with proteins constitute the putative subunits of the OHR complex highest dehalogenating activity (Figure 3). HupL had its in strain BRE15M, and appear to functionally correspond with maximum of relative protein abundance in band VI (179 kDa), the subunits recently described for the OHR complex in D. yet HupLS are detected together in band VII, which prevented mccartyi strain CBDB1. the categorical assumption of an independent hydrogenase After determining protein composition in broad gel bands, module. A remarkable observation was again the colocalization we also determined the presence of proteins within single of the DcpB ortholog with its respective DcpA in band IV (139 defined bands (∼2 mm). For that we ran a sample on a BNE kDa) in a region where HupX and OmeA were absent. gel with proteins solubilized with 1% DDM, stained the gel Finally, a BNE gel lane with proteins extracted with 0.05% with a silver protocol and analyzed dominant protein bands by DDM was analyzed (Figure S5C, Table S21). No RDase nLC−MS/MS (Figure S5A, Table S19). As shown in Figure activity was detected in any of the 19 gel slices picked from the 5A, the RDases (catalytic subunit DcpA, and the RDase with parallel unstained gel. All subunits of the complex described locus tag _160007), the OmeA, the membrane subunit OmeB above, apart from DcpB, were again identified in the excised and the membrane-associated protein HupX were detected in silver stained bands. However, the distribution of the relative the band VII (242 kDa). The membrane anchor DcpB protein abundance across the gel lane pointed to the presence colocalized with DcpA in band VI (202 kDa). The HupLS of three modules (Figure 5C). The two subunits of the Hup complex had its maximum relative abundance in band V (176 complex exhibited identical migration patterns and shared a kDa). To date, it is unknown how protons are pumped across clear maximum relative abundance in band V (123 kDa) the membrane in D. mccartyi strains, but Seidel et al. (2018)11 (Figure 5C), which coincides well with the predicted found that a protein annotated as a K+-insensitive pyrophos- molecular weight of the dimer of HupL/HupS (93.9 kDa). phate energized proton pump (HppA) colocalized with the This observation strengthens the hypothesis that HupL and OmeA/OmeB/HupX module across BNE gels in D. mccartyi HupS form a hydrogenase module also in Dehalogenimonas strain CBDB1. Interestingly, we detected the homologous strains. The DcpA ortholog and the RdhA with locus tag HppA protein (_080016) with a maximum of relative protein _160007 had a similar migration pattern. Unlike the protein 619 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article distribution observed in gels with the 1% DDM or 0.1% DDM growing with different electron acceptors can elucidate solubilized proteins, both RdhA were found in all 19 slices, but whether the RdhA _160007 is specifically induced by 1,2- never more than 20% of relative abundance. Similarly, the DCP or, instead, whether its expression is either broadly putative proton pump protein HppA was broadly distributed induced or even constitutive. Constitutive expression of RdhA across the gel. As noted earlier, RdhB proteins were not in OHRB is common, as illustrated by the dehalogenase detected from 0.05% DDM treatments, suggesting the need of CbdbA80 in D. mccartyi strain CBDB1.36 The colocalization in a higher detergent concentration to detach this protein from the genome as well as in gel slices of these two RdhA may membrane or from the complex. The OmeA/OmeB/HupX suggest either strong interaction, or that they are performing module reached its maximum of relative protein abundance in the same function in different copies of the respiratory band XVIII (450−480 kDa), and they comigrated in the complex. These results are nevertheless intriguing because molecular range between 320 and 480 kDa (bands XV to RdhA _160007 does not have a TAT leader peptide. Further, XIX). This indicated that they were tightly connected to each no RdhA sequences from strain BRE15M contained predicted other. Together, these results indicate weak interaction multiple transmembrane helix domains that might directly between the OmeA/OmeB/HupX module and the HupL/ anchor them to membranes, as was recently described in other HupS module but strong interactions within the modules. The genomes.37−39 One possibility is that both RDases are lack of dehalogenating activity in the different slices of this gel exported together as a heterodimer via the TAT machinery indicates the need of a full multimeric complex to proceed with associated with the DcpA, and once in the outer part of the organohalide respiration, rather than the RdhA catalytic membrane, _160007 is either attached to a different copy of subunit alone. the DcpB or remains attached to DcpA forming a stable Based on these results, we propose a respiratory complex for heterodimer in vivo. This hypothesis would assume that one strain BRE15M that includes subunits of the CISM complex, RdhB protein can anchor different RdhA proteins to the subunits of the Hup hydrogenase, and RDase subunits (Figure membrane. If this is true, the four RdhA proteins without TAT 6). leader peptide encoded in the genome might be anchored to one of the nine encoded RdhB, forming heterodimers with other RdhAs. This may occur even if the RdhA protein without TAT leader peptide is not encoded in the same operons as another RdhA protein. Alternatively, a feasible explanation for the lack of TAT leader peptide is that a RdhA protein might function as a cytoplasmatic nonrespiratory dehalogenase, as reported for aerobic bacteria5,37 and that the association with the OHR complex is an artifact after cell disruption. In this study, we also provide biochemical evidence that RDases in strain BRE15M are part of a large respiratory complex. The nonionic detergent DDM was suitable to detach Figure 6. Model of the OHR complex of D. alkenigignens strain membrane associated proteins while maintaining protein− BRE15M. The proteins found throughout this study to be part of the protein interactions. Protein solubilization with 0.1% (w/v) suspected multimeric respiratory complex are the organohalide DDM enabled the identification of a putative multiprotein respiration involved molybdoenzyme OmeA (locus tag _040002/ complex composed by subunits of the CISM complex, a _040003), and its membrane-anchoring subunit OmeB (_130001), dimeric [NiFe] hydrogenase, and the RDase system in one the hydrogenase large subunit HupL (_070001), and its small subunit single gel slice that was at a position in the gel with maximum HupS (_070002), the HupX (_040001), the RDase DcpA (_160008), and its putative membrane-anchor DcpB (_160009). dechlorination activity within the measured gel lane. Our results with lower DDM concentration (0.05%) were surprisingly counterintuitive because it led to partial ■ DISCUSSION The draft genome sequence of strain BRE15M provides a basis disintegration of the protein complex. This might be explained by the fact that the ionic protein−protein interactions within the complex are not affected by this nonionic detergent, but to study its proteome profile and allowed for a characterization they may disrupt lipid−protein interactions of membrane of its organohalide respiratory system. Annotation of the proteins. In contrast, increasing concentrations of DDM might genome sequence excluded the possibility that organohalide drive the protein subunits to move together, as high salt pushes respiration is quinone-mediated in Dehalogenimonas because hydrophobic interaction together. no functional cytochromes or biosynthetic pathways for The analysis of the complexome using 0.05% (w/v) DDM, quinones were encoded. The genome encoded 31 full-length where the complex partially disintegrates, strongly suggests the rdhA, 22 of which were detected in our proteomic experiments presence of three modules. The largest module comprised (Table S2), even though 1,2-DCP was the only halogenated OmeA, OmeB and HupX. The calculated size of this module of electron acceptor in the medium. The two most abundant 180.9 kDa did not match with the molecular mass RdhA proteins (DcpA ortholog and _160007) are encoded corresponding with the gel slice in which these proteins had together in one operon and were colocalized across the gels its relative maximum abundance (band XVIII, Figure 5C, 450 regardless of the DDM concentration. To date, DcpA was the kDa), suggesting that the Ome/HupX module polymerizes. As only RdhA for which a 1,2-DCP dihaloelimination function observed from the distribution of 0.05% DDM-solubilized was assigned.19 However, in our study the RDase _160007 was proteins across the gel, HupX had a much stronger interaction detected at even higher abundances than DcpA in gels with with OmeA and OmeB than with the two hydrogenase proteins solubilized with 0.1 and 0.05% DDM (Figure S4). subunits, which agrees with previous results with D. mccartyi Further comparative proteomic studies with strain BRE15M strain CBDB1.11 However, a noticeable difference between 620 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article these two strains is that HupX is encoded in an operon in BLASTp search against the amino acid sequence of together with HupL and HupS in D. mccartyi strain CBDB1,10 the formate dehydrogenase β subunit from strain while it forms an operon with OmeA and OmeB in strain BRE15M; ten best hits in BLASTp search against the BRE15M, further supporting its role as the four iron−sulfur amino acid sequence of the protein automatically cluster β-subunit of the CISM module. We also biochemically annotated as polysulfide reductase from strain detected a protein annotated as K+-insensitive pyrophosphate BRE15M; ten best hits in BLASTp search against the energized proton pump (HppA) that migrates similarly to the amino acid sequence of the “protein of unknown OmeA/OmeB/HupX module in a BNE gel with 0.1% DDM- function” (BRE15M_v1_040001) from strain solubilized proteins. However, the broad distribution of HppA BRE15M; protein concentration and total protein across the bands in BNE gels with 0.05% DDM questions any abundances of all of the proteins identified in triplicate strong interaction between HppA and the OmeA/OmeB/ samples of strain BRE15M; ten best hits in BLASTp HupX module but provides a basis to speculate that HppA search against the amino acid sequence of could be part of the respiratory complex of strain BRE15M and BRE15M_v1_160008 from strain BRE15M; propene may translocate protons through the subunits of this complex. production from 1,2-DCP after 24 h in enzymatic assays The hydrogenase subunits HupL and HupS constitute using slices of BNE gels; conditions of BNE experiments another defined module and its facile separation from the other at different DDM concentrations; total protein abun- OHR complex subunits has also been observed in D. mccartyi dances of all of the proteins identified in BNE gel slices strain CBDB1.11 The molecular weight range in which the after bead-beating and solubilization with 0.05%, 0.1%, maximum relative protein abundance of these subunits was and 1%; total protein abundances of all of the proteins detected is 159−179 kDa in the 0.1% DDM treatment (band identified in BNE dominant bands after bead-beating V) and 123−146 kDa in the 0.05% DDM treatment (band V), and solubilization with 0.1%, 0.05%, and 1%; prediction which slightly differs from the theoretical calculated size of of transmembrane helices in putative RdhB protein in 93.9 kDa for this module, however, size determinations in BNE strain BRE15M; arrangement of the DcpA gene and the gels can only considered as rough estimations. Finally, the corresponding B gene, DcpB; number of proteins RDase DcpA, which functioned as terminal reductase in the identified across the length of BNE gels cut into six respiratory complex, is attached to the putative membrane regions after bead-beating and solubilization with anchor DcpB. We detected comigration of both proteins in different % of DDM; absolute abundance of RdhA BNE gels loaded with proteins solubilized with 1% DDM proteins across the length of BNE gels cut into six (band VI, 202 kDa, Figure 5A) and 0.1% DDM (band IV, 139 regions after bead-beating and solubilization with kDa, Figure 5B). These results constitute, together with a different % of DDM; and silver-stained BNE gels with recent study with D. mccartyi strain CBDB1, one of the few protein extracts after bead-beating and solubilization biochemical evidences of specific interaction between an RdhA with different % of DDM (XLSX) and its corresponding RdhB.10 ■ * ASSOCIATED CONTENT sı Supporting Information ■ AUTHOR INFORMATION Corresponding Author The Supporting Information is available free of charge at Ernest Marco-Urrea − Departament d’Enginyeria Quimica, ́ https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00569. Biològica i Ambiental, Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Spain; orcid.org/0000-0002- Metagenomic sequencing and genomic analysis; cell 8033-6553; Phone: +34 935812694; Email: ernest.marco@ harvesting protocol for 1,2-DCP-consuming Dehalogeni- uab.es; Fax: +34935812013 monas cultures; reciprocal BLASTP analyses of protein sequences from D. alkenigignens strains BRE15M and Authors IP3-3; and draft genome annotation details: hydro- ́ genases, formate dehydrogenases, possible organohalide Alba Trueba-Santiso − Departament d’Enginyeria Quimica, respiratory complex, quinones, and cytochromes (PDF) Biològica i Ambiental, Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Spain Genome data for Dehalogenimonas sequenced to date; Kenneth Wasmund − Division of Microbial Ecology, Centre for list and characteristics of RdhAs encoded in the genome Microbiology and Environmental Systems Science, University of of strain BRE15M; characteristics of RdhBs encoded in Vienna, Vienna 1010, Austria the genome of strain BRE15M; ten best hits in BLASTp Jesica M. Soder-Walz − Departament d’Enginyeria Quimica, ́ search against the amino acid sequence of the Biològica i Ambiental, Universitat Autònoma de Barcelona periplasmic [NiFeSe] hydrogenase large subunit from (UAB), Bellaterra 08193, Spain strain BRE15M; ten best hits in BLASTp search against Lorenz Adrian − Department of Environmental Biotechnology, the amino acid sequence of the periplasmic [NiFeSe] Helmholtz Centre for Environmental ResearchUFZ, Leipzig hydrogenase small subunit from strain BRE15M; ten 04318, Germany; Chair of Geobiotechnology, Technische best hits in BLASTp search against the amino acid Universität Berlin, Berlin 10623, Germany; orcid.org/0000- sequence of the formate dehydrogenase, α subunit, from 0001-8205-0842 strain BRE15M; characteristics of the proteins identified in the multiprotein respiratory complex of strain Complete contact information is available at: BRE15M according to TMHMM server and PSORTb; https://pubs.acs.org/10.1021/acs.jproteome.0c00569 ten best hits in BLASTp search against the amino acid sequence of the formate dehydrogenase two subunit α Author Contributions (cytochrome c-553) from strain BRE15M; ten best hits ⊥ A.T.-S. and K.W. contributed equally to this work. 621 https://dx.doi.org/10.1021/acs.jproteome.0c00569 J. Proteome Res. 2021, 20, 613−623 Journal of Proteome Research pubs.acs.org/jpr Article Notes (11) Seidel, K.; Kü hne, J.; Adrian, L. The complexome of Dehalococcoides mccartyi reveals its organohalide respiration-complex The authors declare no competing financial interest. ■ is modular. Front. Microbiol. 2018, 9, 1130. (12) Moe, W. M.; Rainey, F. A.; Yan, J. The genus Dehalogenimonas. ACKNOWLEDGMENTS In Organohalide-Respiring Bacteria; Adrian, L., Löffler, F. E., Eds.; This work has been supported by the Spanish Ministry of Springer Berlin Heidelberg: Berlin, 2016; pp 137−151. Economy and Competitiveness State Research Agency (13) Yang, Y.; Higgins, S. A.; Yan, J.; Ş imşir, B.; Chourey, K.; Iyer, (CTM2016-75587-C2-1-R and PID2019-103989RB-100) co- R.; Hettich, R. L.; Baldwin, B.; Ogles, D. M.; Löffler, F. E. Grape financed by the European Union through the European pomace compost harbors organohalide-respiring Dehalogenimonas Regional Development Fund (ERDF). This work was partly species with novel reductive dehalogenase genes. ISME J. 2017, 11, supported by the Generalitat de Catalunya (Consolidate 2767−2780. (14) Qiao, W.; Luo, F.; Lomheim, L.; Mack, E. E.; Ye, S.; Wu, J.; Research Group 2017-SGR-14). A.T.-S. acknowledges a Edwards, E. A. A dehalogenimonas population respires 1,2,4- predoctoral fellowship from the MINECO (BES-2014- trichlorobenzene and dichlorobenzenes. Environ. Sci. Technol. 2018, 070817) and a German Academic Exchange Service 52, 13391−13398. (DAAD) scholarship (pers. ref. no. 91726320). K.W. is (15) Siddaramappa, S.; Challacombe, J. F.; Delano, S. F.; Green, L. supported by the Austrian Science Fund (FWF) grant no. D.; Daligault, H.; Bruce, D.; Detter, C.; Tapia, R.; Han, S.; Goodwin, P29246-B29. Protein mass spectrometry was done at the L.; Han, J.; Woyke, T.; Pitluck, S.; Pennacchio, L.; Nolan, M.; Land, Centre for Chemical Microscopy (ProVIS) at the Helmholtz M.; Chang, Y.-J.; Kyrpides, N. C.; Ovchinnikova, G.; Hauser, L.; Centre for Environmental Research, which is supported by Lapidus, A.; Yan, J.; Bowman, K. S.; da Costa, M. S.; Rainey, F. A.; European regional development funds (EFRE−Europe Funds Moe, W. M. Complete genome sequence of Dehalogenimonas Saxony), and the Helmholtz Association. The work of L.A. was lykanthroporepellens type strain (BL-DC-9(T)) and comparison to supported by the German Research Council (DFG) within the “Dehalococcoides” strains. Stand. Genomic Sci. 2012, 6, 251−264. Research Group FOR1530 and the SPP1927. We thank the (16) Key, T. A.; Richmond, D. P.; Bowman, K. S.; Cho, Y.-J.; Chun, Biomedical Sequencing Facility (BSF) of Vienna, Austria, for J.; da Costa, M. S.; Rainey, F. A.; Moe, W. M. Genome sequence of the organohalide-respiring Dehalogenimonas alkenigignens type strain DNA sequencing and support. ■ (IP3-3T). Stand. Genomic Sci. 2016, 11, 44. (17) Molenda, O.; Quaile, A. T.; Edwards, E. A. 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