Discovered by Genomics Putative Reductive Dehalogenases with N-Terminus Transmembrane Helixes
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Discovered by genomics putative reductive dehalogenases with N-terminus transmembrane helixes Atashgahi, S. This is a "Post-Print" accepted manuscript, which has been published in "FEMS microbiology ecology" This version is distributed under a non-commercial no derivatives Creative Commons (CC-BY-NC-ND) user license, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and not used for commercial purposes. Further, the restriction applies that if you remix, transform, or build upon the material, you may not distribute the modified material. Please cite this publication as follows: Atashgahi, S. (2019). Discovered by genomics putative reductive dehalogenases with N-terminus transmembrane helixes. FEMS microbiology ecology, 95(5), [fiz048]. https://doi.org/10.1093/femsec/fiz048 1 Discovered by genomics: Putative reductive dehalogenases with N-terminus 2 transmembrane helixes 3 Siavash Atashgahi 1,2,3 4 1 Laboratory of Microbiology, Wageningen University & Research, Wageningen, The 5 Netherlands 6 2 Department of Microbiology, Radboud University, Nijmegen, The Netherlands 7 3 Soehngen Institute of Anaerobic Microbiology, Nijmegen, The Netherlands 8 [email protected]; [email protected]; http://orcid.org/0000-0002-2793- 9 2321; +31243652564 10 11 Abstract 12 Attempts for bioremediation of toxic organohalogens resulted in the identification 13 of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. 14 RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have 15 membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by 16 rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. 17 Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial 18 genome that was not accompanied by an rdhB gene, but contained transmembrane helixes 19 in N-terminus. Therefore, rather than having a separate membrane anchor protein, this 20 putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the 21 membrane with transmembrane helixes. However, functionality of the hybrid putative 22 RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are 23 present in the genomes of pure cultures and uncultured members of Bacteriodetes and 24 Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded 25 hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster 26 phylogenetically separately from the existing RDase groups. With increasing availability of 27 (meta)genomes, more diverse and likely novel rdh genes are expected, but questions 28 regarding their functionality and ecological roles remain open. 1 29 Introduction 30 With the advent of the Industrial Revolution, human impacts on the environment 31 increased dramatically. Hazardous halogenated organic compounds, organohalogens, were 32 widely distributed in the natural environment through careless use and indiscriminate 33 disposal, and caused major public concerns due to possible effects on human and 34 environmental health (Häggblom, 1992). In attempts for organohalogen bioremediation, a 35 hallmark discovery was the identification of microbes that could use organohalogens as 36 electron acceptors and reductively dehalogenate them (Suflita et al., 1982). This new 37 metabolism, later termed organohalide respiration (OHR), has found great practical 38 application in bioremediation. Accordingly, bioaugmentation with microbial consortia 39 containing organohalide-respiring bacteria (OHRB) has become a showcase of successful 40 engineered remediation of contaminated environments (Ellis et al., 2000, Stroo et al., 41 2012). 42 Over the past three decades, a wealth of knowledge has been obtained about the 43 ecophysiology, biochemistry, and environmental distribution of OHRB (Häggblom & 44 Bossert, 2003, Adrian & Löffler, 2016). Using biochemical, PCR-based and (meta)genomic 45 analysis, reductive dehalogenases (RDases) have been identified as the key enzymes of 46 OHR (Lu et al., 2015, Hug, 2016). The RDase-encoding genes (rdh) have a conserved 47 operon structure that consists of rdhA, coding for the catalytic subunit (RdhA); rdhB, coding 48 for a small putative membrane anchor (RdhB) that (presumably) locates the A subunit to 49 the outside of the cytoplasmic membrane; and a variable set of accessory genes (e.g., 50 rdhCTKZED) (Kruse et al., 2016). The catalytic subunits (RdhAs) are characterized by two 51 iron-sulfur clusters (FeS1: CXXCXXCXXXCP; FeS2: CXXCXXXCP) and an N-terminus twin- 52 arginine translocation motif (TAT: RRXFXK) (Holliger et al., 1998). This signal peptide is 53 necessary for secretion of the mature RdhA protein through the cell membrane to the outer 54 side of the cytoplasmic membrane (Smidt & de Vos, 2004). 55 A second type of rdhA genes were discovered that lacked TAT motif, were located 56 in the cytoplasm, and lacked respiratory function. This group was termed as “catabolic” 57 reductive dehalogenase that are used to convert organohalogens to nonhalogenated 2 58 compounds to be used as carbon sources (Chen et al., 2013, Payne et al., 2015). These 59 types of rdhA genes were mostly found in marine than terrestrial environments (Reviewed 60 in Atashgahi et al., 2018). 61 62 Putative rdh genes with N-terminus transmembrane helixes 63 A recent single-cell genomic study from marine sediments in the Aarhus Bay 64 discovered a third type of potential RDases in uncultured Desulfatiglans-related 65 deltaproteobacterium (Jochum et al., 2018). A single-cell genome (SAG2) contained a 66 putative rdh gene that is not accompanied by an rdhB, does not encode a TAT signal 67 peptide, and as a unique feature, encodes three transmembrane helices (TMHs) in the N- 68 terminus. Whereas the known respiratory RDases do not have membrane-integral 69 domains, most RdhBs have three TMHs (Fig. 1). For instance, similar to the RdhB of 70 Desulfitobacterium hafniense Y51 (Fig. 1A), the putative RDase from the uncultured 71 Desulfatiglans-related deltaproteobacterium (Fig. 1B) has an exoplasmic N-terminus, 72 followed by three TMHs. The remaining C-terminus contains the two binding motifs for FeS 73 clusters, features of the known RDases. However, as the possible catalytic site, the C- 74 terminus is facing the inner side of the cytoplasmic membrane (Fig. 1B) which is a likely 75 localization in absence of the TAT signal peptide. The short cytoplasmic loop between helix 76 1 and 2 contains the two conserved glutamic acid residues (EXE motif) (Fig. 1B), proposed 77 to play a role in the RdhA–RdhB interaction (Schubert et al., 2018). Similar cytoplasmic 78 localization of the C-terminus of the putative RDase may enable such an interaction with 79 this loop. Therefore, rather than having a separate membrane anchor protein, this putative 80 RDase is predicted to act like a hybrid of RdhB and RdhA, and likely directly connected to 81 the membrane with the TMHs. 82 The study of Jochum et al further revealed that the hybrid putative rdh is similar to 83 the putative rdh of two deltaproteobacterial pure cultures, i.e., deltaproteobacterium strain 84 NaphS2 and Dethiosulfatarculus sandiegensis (Jochum et al., 2018). Indeed the putative 85 rdh genes of these bacteria are not accompanied by an rdhB gene, lack TAT motif, and 86 contain three N-terminus TMHs. Similar to the putative RDase of the uncultured 3 87 Desulfatiglans-related proteobacterium obtained from the Aarhus Bay (Fig. 1B), the 88 putative RDase of the strain NaphS2 (Fig. 1C) has cytoplasmic C-terminus. In contrast, 89 the putative RDase of D. sandiegensis has exoplasmic C-terminus (Fig. 1D), similar to the 90 known RDases. The EXE motif in the loop between helix 1 and 2 is facing exoplasm, 91 enabling potential interactions with the exoplasmic C-terminus (Fig. 1D). The three 92 putative RDase share 46-58% amino acid identity to each other, but share lower identity 93 to the known RDases, e.g., 26-29% identity to the TceA of Dehalococcoides mccartyi 94 strain195 (DET0079). Although the existence of rdh genes lacking the TAT motif and rdhB 95 were reported in the genomes of strain NaphS2 and D. sandiegensis (Sanford et al., 2016, 96 Liu & Häggblom, 2018), the existence of TMHs in their putative RDase proteins were not 97 reported. However, functionality of the hybrid putative RDases remains unknown. 98 99 The hybrid putative rdh genes are widespread 100 The sequence of the putative RDase of the uncultured proteobacterium obtained 101 from the Aarhus Bay (Jochum et al., 2018) was used as a query in blastp searches against 102 the NCBI non-redundant protein database in December 2018. The results showed that 103 beyond the three identified proteobacterial hybrid putative rdh (Jochum et al., 2018), many 104 other similar genes exist in the genomes of pure cultures as well as metagenome- 105 assembled genomes (MAGs) that have gone unrecognized so far (Table 1). The majority 106 of the sequences have three TMHs (detected using TMHMM Server v. 2.0 (Sonnhammer et 107 al., 1998)), the EXE motifs in their N-terminus, and either cytoplasmic or exoplasmic C- 108 terminus containing the two FeS motifs (Table 1, Fig. 2). The C1-C5 regions from known 109 the RDases are also conserved among the hybrid putative RDases (Fig. S1), however, they 110 are clustered phylogenetically separately from the existing RDase groups (Hug et al., 2013, 111 Hug, 2016) (Fig.