Discovered by Genomics Putative Reductive Dehalogenases with N-Terminus Transmembrane Helixes

Discovered by genomics putative reductive dehalogenases with N-terminus transmembrane helixes

Atashgahi, S.

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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

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.

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

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).

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

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.

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. S2). Notably, the majority of the putative RDases are annotated as

112 hypothetical proteins during automated annotation of the genomes.

113 Of the 11 pure cultures containing hybrid putative rdh in their genomes, eight

114 belong to the Marinilabiliales order within Bacteroidetes, that have been isolated from

115 water or sediment samples in marine environment (Table 1). Among these, three strains

116 belong to the genus Marinifilum, Gram-negative facultative anaerobes that can tolerate

117 moderate salt concentrations (Na et al., 2009, Ruvira et al., 2013, Fu et al., 2018).

118 Interestingly, hybrid putative rdh genes were also found in the MAGs of uncultured

119 Marinilabiliales obtained from perchlorate-reducing enrichment cultures originating from

120 marine sediments (Barnum et al., 2018). These genomes mostly lacked respiratory

121 perchlorate, chlorate, oxygen, and sulfur reductases and were proposed to be specialized

122 for the fermentation of dead cells (Barnum et al., 2018). These finding indicate an

123 important role of the hybrid putative rdh genes in Marinilabiliales members. Another pure

124 culture harbouring the hybrid putative rdh in its genome is Caldithrix abyssi, a thermophilic

125 anaerobic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent (Miroshnichenko

126 et al., 2003). Calditrichaeota are abundant seabed microbes with genomic potential to

127 degrade detrital proteins through the use of extracellular peptidases (Marshall et al.,

128 2017).

129 Except the MAGs obtained from the marine perchlorate-reducing enrichment

130 cultures (Barnum et al., 2018), all other MAGs containing hybrid putative rdh were

131 obtained from harsh environments such as hydrothermal vents (Dombrowski et al., 2017,

132 Dombrowski et al., 2018), hot springs (Wilkins et al., 2018), wetlands with extremely high

133 concentrations of dissolved organic carbon and diverse sulfur species (Martins et al., 2018),

134 deep terrestrial environments (Hernsdorf et al., 2017, Momper et al., 2017), etc (Table 1).

135 Most of the sequences from the MAGs were obtained from hydrothermal vent sediments in

136 Guaymas Basin (Gulf of California) with fluctuating temperature and chemical gradients

137 (Dombrowski et al., 2017, Dombrowski et al., 2018). The MAGs are mostly from uncultured

138 Bacteriodetes and Deltaproteobacteria, but also from the candidate divisions (Table 1).

139 Members of all these phyla have known/proposed diverse metabolic potential, and may

140 not be restricted to reductive dehalogenation. However, physiological proofs for OHR have

141 only been obtained for deltaproteobacterial members with classic rdh gene operon i.e.

142 rdhA, rdhB and one or more transcriptional regulatory genes (Sanford et al., 2016, Liu &

143 Häggblom, 2018).

144

145 Outstanding questions

146 Genomics and allied technologies have greatly increased the diversity of putative

147 rdh genes in recent years, and extended their distribution from contaminated environments

148 to deep subsurface (Table 1), Antarctic soils (Zlamal et al., 2017), and even human and

149 animal intestinal tract (Atashgahi et al., 2018). With the expanding availability of the

150 bacterial genomes and increasing application of deep sequencing in diverse environments,

151 much more diverse and likely novel rdh genes are expected in future. This brings forward

152 major open questions:

153 - Do the newly discovered genes encode RDases? If they indeed encode RDases,

154 what are their functions? Three roles have been shown for the known RDases:

155 energy conservation by OHR, and facilitated fermentation of organic substrates

156 (e.g. pyruvate, lactate, or yeast extract) by reoxidation of respiratory cofactors

157 for membrane-bound RDases, and catabolic reductive dehalogenation for

158 cytoplasmic RDases (Fincker & Spormann, 2017). Can the hybrid putative

159 RDases with cytoplasmic C-terminus be involved in catabolic reductive

160 dehalogenation, facilitated fermentation, or both? In turn, how are the hybrid

161 putative RDases with exoplasmic C-terminus secreted through the cell

162 membrane in absence of TAT signal peptide?

163 - If indeed involved in reductive dehalogenation, what are the physiological

164 organohalogen substrates of the hybrid putative RDases? The lack of correlation

165 between the rdh sequences and their organohalogen substrates has precluded

166 the ability to predict substrates for novel genes, and to test their functionality

167 using the predicted organohalogens.

168 - Why the majority of the environmental hybrid putative rdh sequences and rdh-

169 containing pure cultures have been obtained from harsh environments? Can it

170 be that their physiological organohalogen substrates are found in these

171 environments?

172 - What are the ecological functions of the microbes containing (the hybrid

173 putative) RDases? Detoxification of organohalogens and thereby securing a

174 hospitable environments for themselves and the nearby organisms? Providing

175 carbon sources for themselves (catabolic RDase) or others (respiratory RDase)?

176 - Can (the hybrid putative) RDases be involved in the production of halogenated

177 bioactive compounds as was shown for biosynthesis of marine bacterial pyrroles

178 mediated by a reductive debrominase that utilizes a redox thiol mechanism (El

179 Gamal et al., 2016)? Likewise, can the RDases participate in in the production

180 of halogenated bioactive compounds in Eukaryotes such as sponges that are

181 known to harbour Deltaproteobacteria with rdh genes (Wilson et al., 2014, Liu

182 et al., 2017)?

183

184 Funding

185 This work was supported by the SIAM Gravitation grant “Microbes for Health and the

186 Environment” (Project 024.002.002) of the Netherlands Ministry of Education, Culture and

187 Science, and the Netherlands Science Foundation (NWO).

188

189 Acknowledgements

190 I thank Kasper U. Kjeldsen for providing sequence information, Torsten Schubert and Peng

191 Peng for assistance in creating figures, and two anonymous reviewers for their constructive

192 comments.

193

194 Conflict of interest

195 None declared.

196

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320

321 Figure and Table Legends

322 Figure 1. Predicted topology of the PceB protein of D. hafniense Y51 (A) and N-terminus

323 TMHs of the hybrid putative RDases from uncultured deltaproteobacterium obtained from

324 the Aarhus Bay (B), deltaproteobacterium strain NaphS2 (C) and D. sandiegensis (D). The

325 position of the EXE motif is indicated by a star. Note that in panel B, C, and D, only partial

326 sequences of the hybrid putative RDases containing N-terminus TMHs were shown. TMHs

327 were detected using TMHMM Server v. 2.0 (Sonnhammer et al., 1998). Permission to

328 reprint panel A was obtained from (Schubert et al., 2018).

329 Figure 2. Sequence alignment of the hybrid putative RDases. Only conserved sequence

330 motifs among experimentally characterized RDases (TAT, FeS1, FeS2), and the conserved

331 glutamic acid residues (EXE) are included. The accession numbers are ordered according

332 to Table 1, except the first accession number that belongs to TceA of Dehalococcoides

333 mccartyi strain 195. ClustalW (Thompson et al., 1994) multiple sequence alignment was

334 conducted using BioEdit version 7.2.5 (http:/bioedit.software.informer.com/).

335 Table 1. List of the hybrid putative RDases with TMHs in their N-terminus. Sequence

336 information and the predicted functions by the automated annotation for each sequence

337 are included in Supporting Information.

338 Figure 1

339

340 Figure 2

341

Organism Length TMH C-terminus GenBank Sample source used for Reference (aa) orientation accession (meta)genome sequencing number Deltaproteobacteria bacterium 482 3 Cytoplasmic - a Marine sediment from Aarhus Bay (Jochum et al., 2018) Dethiosulfatarculus sandiegensis 487 3 Exoplasmic WP_082464279 Pure deltaproteobacterial culture isolated (Davidova et al., 2016) from a methanogenic long-chain paraffins degrading consortium obtained from marine sediments Deltaproteobacterium NaphS2 478 3 Cytoplasmic EFK11122 Pure deltaproteobacterial culture isolated (Galushko et al., 1999, from naphthalene-degrading enrichment Didonato Jr et al., 2010) obtained from marine sediments Marinifilaceae bacterium strain SPP2 459 3 Exoplasmic WP_096429615 Pure Marinilabiliales culture isolated from (Watanabe et al., 2018) the Antarctic marine sediment Marinifilum fragile 456 3 Exoplasmic WP_054715848 Pure Marinilabiliales culture isolated from (Na et al., 2009) tidal flat sediment in Korea Marinifilum breve 457 3 Cytoplasmic WP_110360576 Pure Marinilabiliales culture isolated from (Fu et al., 2018) the Yongle Blue Hole in the South China Sea Marinifilum flexuosum 454 3 Cytoplasmic WP_120240634 Pure Marinilabiliales culture isolated from (Ruvira et al., 2013) coastal Mediterranean Sea water Ancylomarina sp. M1P 450 3 Cytoplasmic WP_125029802 Pure Marinilabiliales culture isolated from Unpublished Black Sea water Labilibaculum filiforme 454 3 Exoplasmic WP_101260201 Pure Marinilabiliales culture isolated from (Vandieken et al., 2018) the subsurface sediments of the Baltic Sea Labilibacter marinus 444 3 Cytoplasmic WP_066632432 Pure Marinilabiliales culture isolated from (Liu et al., 2015, Lu et al., marine sediment at Weihai in China 2017) Salinivirga cyanobacteriivorans 453 3 Cytoplasmic WP_057954221 Pure Marinilabiliales culture isolated from (Ben Hania et al., 2017) the suboxic zone of a hypersaline cyanobacterial mat Caldithrix abyssi 444 3 Cytoplasmic WP_006930498 Pure Calditrichales culture isolated from (Miroshnichenko et al., Mid-Atlantic Ridge hydrothermal vent 2003, Kublanov et al., 2017) Deltaproteobacteria bacterium 491 3 Exoplasmic RLB29679.1 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 451 3 Exoplasmic RLB34449 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 455 3 Exoplasmic RLC06278 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 456 3 Exoplasmic RLB93792 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 455 3 Exoplasmic RLC22838 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 414 3 Cytoplasmic RLC21098 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 497 3 Cytoplasmic RLB22016 Hydrothermal sediments (Dombrowski et al., 2018)

Deltaproteobacteria bacterium 359 3 Cytoplasmic RLC02598 Hydrothermal sediments (Dombrowski et al., 2018) Desulfobacteraceae bacterium 478 3 Exoplasmic OQY12990 Hydrothermal sediment (Dombrowski et al., 2017) 4572_187 Desulfobacteraceae bacterium 454 3 Exoplasmic OQY53460 Hydrothermal sediments (Dombrowski et al., 2017) 4572_89 Bacteroidetes bacterium 457 3 Exoplasmic RLD45891 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 447 3 Exoplasmic RLD65038 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 457 3 Exoplasmic RLD32997 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 402 1 Exoplasmic RLD55593 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 469 3 Cytoplasmic RLD42118 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 4484_249 446 2 Cytoplasmic OQX80664 Hydrothermal sediments (Dombrowski et al., 2017) Bacteroidetes bacterium 476 4 Cytoplasmic RLD38167 Hydrothermal sediments (Dombrowski et al., 2018) Bacteroidetes bacterium 454 3 Cytoplasmic RLD75418 Hydrothermal sediments (Dombrowski et al., 2018) Acidobacteria bacterium 450 3 Cytoplasmic RLE20106 Hydrothermal sediments (Dombrowski et al., 2018) Chloroflexi bacterium 453 3 Exoplasmic RLD03862 Hydrothermal sediments (Dombrowski et al., 2018) Chloroflexi bacterium 457 3 Exoplasmic RLD00869 Hydrothermal sediments (Dombrowski et al., 2018) Chloroflexi bacterium 453 3 Cytoplasmic RLD11393 Hydrothermal sediments (Dombrowski et al., 2018) Bacterium 453 3 Cytoplasmic RKZ14043 Hydrothermal sediments (Dombrowski et al., 2018) Bacterium 448 3 Cytoplasmic RKZ19839 Hydrothermal sediments (Dombrowski et al., 2018) Candidate division KSB1 bacterium 457 3 Exoplasmic RKY76530 Hydrothermal sediments (Dombrowski et al., 2018) Candidate division KSB1 bacterium 417 1 Exoplasmic OQX94610 Hydrothermal sediments (Dombrowski et al., 2017) 4572_119 Candidate division KSB1 bacterium 458 3 Exoplasmic OQX85480 Hydrothermal sediments (Dombrowski et al., 2017) 4484_87 Candidate division Zixibacteria 501 3 Exoplasmic RKX26209 Hydrothermal sediments (Dombrowski et al., 2018) bacterium Candidate division Zixibacteria 461 3 Cytoplasmic RKX27199 Hydrothermal sediments (Dombrowski et al., 2018) bacterium Candidatus Aminicenantes 511 3 Cytoplasmic OQX52307 Hydrothermal sediments (Dombrowski et al., 2017) bacterium 4484_214 Candidatus Aminicenantes 469 3 Cytoplasmic RLE02852 Hydrothermal sediments (Dombrowski et al., 2018) bacterium Candidatus Omnitrophica bacterium 389 1 Exoplasmic RKY41132 Hydrothermal sediments (Dombrowski et al., 2018) Deltaproteobacteria bacterium 463 3 Exoplasmic PLX41189 Perchlorate-reducing communities (Barnum et al., 2018) Salinivirgaceae bacterium 497 4 Exoplasmic PLX17815 Perchlorate-reducing communities (Barnum et al., 2018) Marinilabiliales bacterium 456 3 Exoplasmic PLW95329 Perchlorate-reducing communities (Barnum et al., 2018) Marinilabiliales bacterium 446 3 Exoplasmic PLW99613 Perchlorate-reducing communities (Barnum et al., 2018) Marinilabiliales bacterium 455 3 Cytoplasmic PLW92978 Perchlorate-reducing communities (Barnum et al., 2018) Marinilabiliales bacterium 454 3 Cytoplasmic PLX09622 Perchlorate-reducing communities (Barnum et al., 2018)

Marinilabiliales bacterium 458 3 Cytoplasmic PLX19442 Perchlorate-reducing communities (Barnum et al., 2018) Marinilabiliales bacterium 452 3 Cytoplasmic PLX02242 Perchlorate-reducing communities (Barnum et al., 2018) Bacteroidetes bacterium 432 2 Exoplasmic OFX85901 Aquifers (Anantharaman et al., 2016) GWE2_32_14 Bacteroidetes bacterium 462 3 Exoplasmic OFX81662 Aquifers (Anantharaman et al., 2016) GWE2_40_15 Candidatus Fischerbacteria 447 3 Cytoplasmic OGF65237 Aquifers (Anantharaman et al., 2016) bacterium RBG_13_37_8 Desulfobacterales bacterium 456 3 Cytoplasmic OGR28476 Aquifers (Anantharaman et al., 2016) RIFOXYA12_FULL_46_15 Desulfobacteraceae bacterium 476 3 Exoplasmic RPI80002 Wetlands (Martins et al., 2018) Deltaproteobacteria bacterium 468 3 Exoplasmic RPJ06807 Wetlands (Martins et al., 2018) Bacteroidales bacterium 454 3 Exoplasmic RPH31952 Wetlands (Martins et al., 2018) Bacterium SM23_31 446 3 Cytoplasmic KPK88368 Estuary sediments (Baker et al., 2015) Candidate division Zixibacteria 441 3 Cytoplasmic KPL04245 Estuary sediments (Baker et al., 2015) bacterium SM23_73_2 Latescibacteria bacterium DG_63 453 3 Cytoplasmic KPJ61247 Estuary sediments (Baker et al., 2015) Deltaproteobacteria bacterium HGW- 542 3 Exoplasmic PKN64391 Deep terrestrial subsurface sediments (Hernsdorf et al., 2017) Deltaproteobacteria-15 Candidate division Zixibacteria 459 3 Exoplasmic PKK82132 Deep terrestrial subsurface sediments (Hernsdorf et al., 2017) bacterium HGW-Zixibacteria-1 Desulfobacteraceae bacterium 489 3 Cytoplasmic RJR39500 Deep terrestrial subsurface fluids (Momper et al., 2017) Marinimicrobia bacterium 46_43 453 3 Exoplasmic KUK91590 Oil Reservoirs (Hu et al., 2016) Candidatus Korarchaeota archaeon 452 3 Exoplasmic PMB78244 Hot springs (Wilkins et al., 2018) Desulfobacterales bacterium 488 3 Exoplasmic OEU64681 Marine sediments Unpublished S5133MH16 Candidate division KSB1 bacterium 432 3 Cytoplasmic RQW00415 - b Unpublished a Not available; sequence information provided in supporting information b Not available

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