Peatland Acidobacteria with a Dissimilatory Sulfur Metabolism

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bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Peatland Acidobacteria

2 with a dissimilatory sulfur metabolism

3 Bela Hausmann1,2, Claus Pelikan1, Craig W. Herbold1, Stephan Köstlbacher1, Mads Albertsen3, 4 Stephanie A. Eichorst1, Tijana Glavina del Rio4, Martin Huemer1, Per H. Nielsen3, Thomas 5 Rattei5, Ulrich Stingl6, Susannah G. Tringe4, Daniela Trojan1, Cecilia Wentrup1, Dagmar 6 Woebken1, Michael Pester2,7, and Alexander Loy1

7 1 University of Vienna, Research Network Chemistry meets Microbiology, Department of 8 Microbiology and Ecosystem Science, Division of Microbial Ecology, Vienna, Austria

9 2 University of Konstanz, Department of Biology, Konstanz, Germany

10 3 Aalborg University, Department of Chemistry and Bioscience, Center for Microbial 11 Communities, Aalborg, Denmark

12 4 US Department of Energy Joint Genome Institute, Walnut Creek, CA, USA

13 5 University of Vienna, Research Network Chemistry meets Microbiology, Department of 14 Microbiology and Ecosystem Science, Division of Computational Systems Biology, Vienna, 15 Austria

16 6 University of Florida, Fort Lauderdale Research and Education Center, UF/IFAS, Department for 17 Microbiology and Cell Science, Davie, FL, USA

18 7 Leibniz Institute DSMZ, Braunschweig, Germany

19 Correspondence: Michael Pester, Leibniz Institute DSMZ, Inhoffenstraße 7B, 38124 20 Braunschweig, Germany. Phone: +49 531 2616 237. Fax: +49 531 2616 418. E-mail: 21 [email protected]

22 Abstract 23 Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and 24 consequently greenhouse gas emissions from these globally relevant environments. However, 25 their identities and physiological properties are largely unknown. By applying a functional 26 metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel 27 Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, 28 dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes 29 also encoded dsrL, a unique gene of the sulfur oxidation pathway. Metatranscriptome analysis 30 demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their 31 upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, 32 which, however, could also operate in reverse for sulfur oxidation as recently shown for other 33 microorganisms. Acidobacteria that only harbored genes for sulfite reduction additionally 34 encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur 35 compounds as complementary energy sources. Further metabolic potentials included 36 polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative 37 capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and 38 genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a 39 DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic 40 Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for 41 energy conservation.

42 Introduction 43 Specialized microorganisms oxidize, reduce, or disproportionate sulfur compounds of various 44 oxidation states (–II to +VI) to generate energy for cellular activity and growth and thereby drive 45 the global sulfur cycle. The capability for characteristic sulfur redox reactions such as 46 dissimilatory sulfate reduction or sulfide oxidation is not confined to single taxa but distributed 47 across different, often unrelated taxa. The true extent of the taxon-diversity within the different 48 guilds of sulfur microorganisms is unknown (Wasmund et al., 2017). However, ecological studies 49 employing specific sulfur metabolism genes (e.g., dissimilatory adenylyl-sulfate reductase- 50 encoding aprBA, dissimilatory sulfite reductase-encoding dsrAB, or soxB that codes for a part of 51 the thiosulfate-oxidizing Sox enzyme machinery) as phylogenetic and functional markers have 52 repeatedly demonstrated that only a minor fraction of the sulfur metabolism gene diversity in 53 many environments can be linked to recognized taxa (Meyer et al., 2007; Müller et al., 2015; 54 Watanabe et al., 2016). A systematic review of dsrAB diversity has revealed that the reductive 55 bacterial-type enzyme branch of the DsrAB tree contains at least thirteen family-level lineages 56 without any cultivated representatives. This indicates that major taxa of sulfate-/sulfite-reducing 57 microorganisms have not yet been identified (Müller et al., 2015).

58 Wetlands are among those ecosystems that harbor a diverse community of microorganisms with 59 reductive-type DsrAB, most of which cannot be identified because they are distant from 60 recognized taxa (Pester et al., 2012). Sulfur-cycling microorganisms provide significant 61 ecosystem services in natural and anthropogenic wetlands, which are major sources of the 62 climate-warming greenhouse gas methane (Kirschke et al., 2013; Saunois et al., 2016). While 63 inorganic sulfur compounds are often detected only at low concentration (lower µM range), fast 64 sulfur cycling nevertheless ensures that oxidized sulfur compounds such as sulfate are rapidly 65 replenished for anaerobic respiration. The activity of sulfate-reducing microorganisms (SRM) 66 fluctuates with time and space, but at peak times can account for up to 50% of anaerobic 67 mineralization of organic carbon in wetlands (Pester et al., 2012). Simultaneously, SRM prevent 68 methane production by rerouting carbon flow away from methanogenic archaea. Autochthonous 69 peat microorganisms that are affiliated to known SRM taxa, such asDesulfosporosinus, 70 Desulfomonile, and Syntrophobacter, are typically low abundant (Loy et al., 2004; Costello and 71 Schmidt, 2006; Dedysh et al., 2006; Kraigher et al., 2006; Pester et al., 2010; Steger et al., 72 2011; Tveit et al., 2013; Hausmann et al., 2016). In contrast, some microorganisms that belong 73 to novel, environmental dsrAB lineages can be considerably more abundant in wetlands than 74 species-level dsrAB operational taxonomic units of known taxa (Steger et al., 2011). However, 75 the taxonomic identity of these novel dsrAB-containing microorganisms and their role in sulfur 76 and carbon cycling has yet to be revealed.

3 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 77 Here, we recovered thirteen metagenome-assembled genomes (MAGs) encoding DsrAB of 78 uncultured species from a peat soil by using a targeted, functional metagenomics approach. We 79 analyzed expression of predicted physiological capabilities of the MAGs by metatranscriptome 80 analyses of anoxic peat soil microcosms that were periodically stimulated by small additions of 81 individual fermentation products with or without supplemented sulfate (Hausmann et al., 2016). 82 Our results suggest that some facultatively anaerobic members of the diverse Acidobacteria 83 community in wetlands employ a dissimilatory sulfur metabolism.

84 Materials and methods

85 Anoxic microcosm experiments, stable isotope probing, and nucleic acids isolation 86 DNA and RNA samples were retrieved from a previous peat soil microcosm experiment 87 (Hausmann et al., 2016). Briefly, soil from 10–20 cm depth was sampled from an acidic peatland 88 (Schlöppnerbrunnen II, Germany) in September 2010, and stored at 4 °C for one week prior to 89 nucleic acids extractions and set-up of soil slurry incubations. Individual soil slurry microcosms 90 were incubated anoxically (100% N₂ atmosphere) in the dark at 14 °C, and regularly amended 91 with low amounts (<200 µM) of either formate, acetate, propionate, lactate, butyrate, or without 92 any additional carbon sources (six replicates each). In addition, half of the microcosms for each 93 substrate were periodically supplemented with low amounts of sulfate (initial amendment of 94 190–387 µM with periodic additions of 79–161 µM final concentrations). DNA and RNA were 95 extracted from the native soil and RNA was additionally extracted from the soil slurries after 8 96 and 36 days of incubation.

97 Furthermore, DNA was obtained from the heavy, ¹³C-enriched DNA fractions of a previous DNA- 98 stable isotope probing (DNA-SIP) experiment with soil from the same site (Pester et al., 2010). 99 Analogous to the single-substrate incubations, anoxic soil slurries were incubated for two 100 months with low-amounts of sulfate and a ¹³C-labelled mixture of formate, acetate, propionate, 101 and lactate. DNA was extracted, separated on eight replicated density gradients, and DNA from 102 a total of 16 heavy fractions (density 1.715–1.726 g mL⁻¹) was pooled for sequencing.

103 Additional DNA was obtained from soils that were sampled from different depths in the years 104 2004 and 2007 (Steger et al., 2011).

105 Quantitative PCR and metagenome/-transcriptome sequencing 106 Abundances of Acidobacteria subdivision 1, 2, and 3 in soil samples from different years and 107 depths were determined by newly-developed 16S rRNA gene-targeted real-time quantitative 108 PCR (qPCR) assays (Supplementary Methods). Native soil DNA (two libraries), heavy 13C- 109 enriched DNA (three libraries), and native soil RNA, and RNA samples from the microcosms were 110 sequenced on an Illumina HiSeq2000 system (Supplementary Methods).

4 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 111 Binning, phylogeny, and annotation of DsrAB-encoding genomes 112 The differential coverage binning approach by Albertsen et al. (2013) was applied to extract 113 MAGs of interest. The raw FASTQ paired-end reads were imported into the CLC Genomics 114 Workbench 5.5.1 (CLC Bio) and trimmed using a minimum Phred quality score of 20 with no 115 ambiguous nucleotides allowed. TruSeq adapters were removed and a minimum length filter of 116 50 nt was applied. This resulted in 214, 171, 233, 49, and 294 million reads after quality filtering 117 and trimming for the two native soil and three SIP metagenomes, respectively (84–95% of the 118 raw reads). All reads were co-assembled using CLCs de novo assembly algorithm (kmer size 63, 119 bubble size 50, minimum scaffold size 1000 nt). The reads from all five metagenomes were 120 independently mapped to the assembled scaffolds using CLCs map to reference function 121 (minimum length 0.7, minimum similarity 0.95) to obtained the scaffold coverage. The SIP 122 metagenomes were merged into one mapping. 137, 112, and 376 million reads could be 123 mapped to the two native soil metagenomes and the SIP metagenome, respectively (64–66% of 124 quality filtered reads). Gene prediction of the complete assembly was performed using prodigal 125 (Hyatt et al., 2010). In addition to the detection and taxonomic classification of 105 essential 126 marker genes (Albertsen et al., 2013), dsrA and dsrB homologs were identified using TIGRFAM’s 127 hidden Markov model (HMM) profiles TIGR02064 and TIGR02066, respectively, with HMMER 3.1 128 (Eddy, 2011) and the provided trusted cut-offs. Additional dsrAB-containing scaffolds were 129 identified by using tblastn with the published DsrAB database as a query against the assembly 130 (Müller et al., 2015). DsrAB sequences were classified by phylogenetic analysis (Supplementary 131 Methods; Müller et al., 2015). Binning and decontamination was finalized utilizing the G+C 132 content and tetramer frequencies of the scaffolds, as well as paired-end information, as 133 described and recommended in Albertsen et al. (2013). Completeness, contamination, and 134 strain heterogeneity was estimated using CheckM 1.0.6 (Parks et al., 2015) with lineage-specific 135 marker sets selected at phylum rank (or class rank for Proteobacteria). MAGs were 136 taxonomically classified by phylogenomic analysis of concatenated marker sequences and 137 calculation of average nucleic and amino acid identities (ANI, AAI, Supplementary Methods). 138 MAGs were annotated using MaGe (Vallenet et al., 2017) and eggNOG (Huerta-Cepas et al., 139 2016). Genes of interest (Supplementary Table S2) were manually curated using the full range 140 of tools integrated in MaGe (Supplementary Methods).

141 Genome-centric activity analysis: iRep and metatranscriptomics 142 The index of replication (iRep) was calculated for each MAG with the combined native soil 143 metagenomes. Settings and thresholds were applied as recommended (Brown et al., 2016) 144 using bowtie2 (Langmead and Salzberg, 2012) and the iRep script with default settings. Quality- 145 filtered metatranscriptome reads were mapped to all genomes using bowtie2 and counted with 146 featureCounts (Liao et al., 2014). To determine gene expression changes, we applied the 147 DESeq2 pipeline with recommended settings (Love et al., 2014) (Supplementary Methods).

153 Results

154 Functional metagenomics: Recovery of dsrAB-containing acidobacterial genomes 155 from native soil and ¹³C-DNA fraction metagenomes 156 This study was conducted with soil samples from the Schlöppnerbrunnen II peatland in 157 Germany, which is a long-term study site with active sulfur cycling and harbors a large diversity 158 of unknown microorganisms with divergent dsrAB genes (Steger et al., 2011; Pester et al., 159 2012). We initially generated co-assembled metagenomes from native peat soil DNA (53 Gb) 160 and a pool of DNA extracts from the heavy fractions of a previous DNA-stable isotope probing 161 (DNA-SIP) experiment with soil from the same peat (101 Gb). The heavy fractions, which were 162 obtained from anoxic peat incubations with periodically supplemented sulfate and a mixture of 163 ¹³C-labelled formate, acetate, propionate, and lactate at low concentrations, were enriched in 164 DNA from Desulfosporosinus and also harbored DNA from yet unidentified dsrAB-containing 165 microorganisms (Pester et al., 2010). Based on the metagenome data, the native peat was 166 dominated by Acidobacteria (61%), but also had Actinobacteria, Alphaproteobacteria, and 167 Deltaproteobacteria as abundant (>5%) phyla/classes (Figure 1). Dominance of Acidobacteria, 168 Alpha- and Deltaproteobacteria is typical for peatlands (Dedysh, 2011). Quantitative PCR 169 confirmed that Acidobacteria subdivisions 1, 2, and 3 persistently dominated the 170 Schlöppnerbrunnen II peat microbiota in oxic and anoxic soil layers (Supplementary Methods, 171 Figure 1), as observed in other peatlands (Serkebaeva et al., 2013; Urbanová and Bárta, 2014; 172 Ivanova et al., 2016).

173 We identified 36 complete or partial dsrAB genes on scaffolds of the composite metagenome 174 and subsequently recovered thirteen MAGs of DsrAB-encoding bacteria by differential coverage 175 binning (Supplementary Table S1, Albertsen et al., 2013). Twenty-eight dsrAB sequences were 176 part of the reductive bacterial-type DsrAB family branch and were closely related to previously 177 recovered sequences from this and other wetlands (Supplementary Figure S1). These dsrAB 178 sequences were affiliated to the known SRM genera Desulfosporosinus (Firmicutes, n=1, one 179 MAG) and Syntrophobacter (Deltaproteobacteria, n=3, two MAGs), the Desulfobacca 180 acetoxidans lineage (n=1), and the uncultured DsrAB family-level lineages 8 (n=19, seven 181 MAGs) and 10 (n=4). Six sequences grouped with the oxidative bacterial-type DsrAB family and 182 were distantly affiliated with Sideroxydans lithotrophicus (Betaproteobacteria, n=5, two MAGs) 183 or Rhodomicrobium vannielii (Alphaproteobacteria, n=1) (Supplementary Figure S2).

6 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 184 Interestingly, two of our sequences (n=2, one MAG) and a DsrAB sequence from the candidate 185 phylum Rokubacteria (Hug et al., 2016) formed a completely novel basal lineage outside the 186 four previously recognized DsrAB enzyme families (Supplementary Figure S2) (Müller et al., 187 2015). The thirteen partial to near complete dsrAB-containing MAGs had moderate to no 188 detectable contamination as assessed by CheckM (Supplementary Table S1) (Parks et al., 2015) 189 and derived from Acidobacteria subdivisions 1 and 3 (SbA1–7), Desulfosporosinus (SbF1), 190 Syntrophobacter (SbD1, SbD2), Betaproteobacteria (SbB1, SbB2), and Verrucomicrobia (SbV1), 191 as inferred by phylogenetic analysis of DsrAB sequences (Supplementary Figures S1 and S2) 192 and concatenated sequences of single-copy, phylogenetic marker genes (Supplementary Figure 193 S3). Only the Desulfosporosinus and Syntrophobacter MAGs contained rRNA gene sequences.

194 Phylogenomic analysis showed that Acidobacteria MAGs SbA1, SbA5, and SbA7 are affiliated 195 with subdivision 1, while SbA3, SbA4, and SbA6 are affiliated with subdivision 3 (Supplementary 196 Figure S3). The partial MAG SbA2 lacked the marker genes used for phylogenomic treeing, but 197 was unambiguously assigned to Acidobacteria using an extended marker gene set (Albertsen et 198 al., 2013) and DsrAB phylogeny. The two near complete (96%) MAGs SbA1 and SbA5 have a size 199 of 5.4 and 5.3 Mb, respectively. The G+C content of all MAGs ranges from 58% to 63% 200 (Supplementary Table S1). This in accordance with genome characteristics of acidobacterial 201 isolates, which have genome sizes of 4.1–10.0 Mb and G+C contents of 57–62% (Ward et al., 202 2009; Rawat et al., 2012,). SbA1 and SbA7 form a monophyletic clade in the Acidobacteria 203 subdivision 1 with an AAI (Rodriguez-R and Konstantinidis, 2014) of 63% (Supplementary Figure 204 S3) and DsrAB identity of 80%. They have 56% AAI to their closest relative, Ca. Koribacter 205 versatilis, which is lower than AAIs among members of known acidobacterial genera (60–71%). 206 The third MAG from subdivision 1, SbA5, is affiliated with Terracidiphilus gabretensis with an AAI 207 of 61%. DsrAB identity of SbA5 to SbA1 and SbA7 is 79%. The three subdivision 3 MAGs form a 208 monophyletic clade with Ca. Solibacter usitatus (Supplementary Figure S3). SbA3, SbA4, and 209 SbA6 have AAIs of 59–73% amongst them and 61–62% to Ca. S. usitatus. DsrAB identity 210 amongst the three MAGs is 80–94% and 74–79% to the subdivisions 1 MAGs.

211 The DsrAB sequences encoded on all seven MAGs are affiliated with the uncultured DsrAB 212 family-level lineage 8 (Supplementary Figure S1), which so far only consisted of environmental 213 dsrAB sequences of unknown taxonomic identity (Müller et al., 2015). Based on these MAGs and 214 metatranscriptome analyses of anoxic peat soil microcosms, we here describe the putative 215 metabolic capabilities of these novel DsrAB-encoding Acidobacteria. Details on the other MAGs 216 will be described elsewhere (Hausmann et al., unpublished; Anantharaman et al., unpublished). 217 Functional interpretations of the recovered MAGs are made under the premise that the genomes 218 are not closed, and thus it is unknown if genes are absent in these organisms or are missing due 219 to incomplete sequencing, assembly, or binning.

7 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 220 Dissimilatory sulfur metabolism 221 Although Acidobacteria are abundant in diverse environments with active sulfur cycling 222 (Serkebaeva et al., 2013; Urbanová and Bárta, 2014; Sánchez-Andrea et al., 2011; Wang et al., 223 2012), this is the first discovery of members of this phylum with a putative dissimilatory sulfur 224 metabolism. SbA2, SbA3, and SbA7 encode the complete canonical pathway for dissimilatory 225 sulfate reduction, including homologs for sulfate transport (sulP and/or dass, not in SbA7) and 226 activation (sat, ppa, hppA), adenosine 5′-phosphosulfate (APS) reduction (aprBA, qmoABC), and 227 sulfite reduction (dsrAB, dsrC, dsrMKJOP) (Figure 2, Supplementary Table S2a) (Santos et al., 228 2015). SbA1, SbA4, SbA5, and SBA6 have an incomplete sulfate reduction gene set but contain 229 all dsr genes for sulfite reduction. Several other dsr genes were present on some of the MAGs. 230 The dsrD and dsrN genes occurred in pairs. The role of the small DsrD protein is unresolved, but 231 its ubiquity among SRM suggests an essential function in sulfate reduction (Hittel and 232 Voordouw, 2000). DsrN is a homolog of cobyrinate a,c-diamide synthase in cobalamin 233 biosynthesis and may be involved in amidation of the siroheme prosthetic group of DsrAB 234 (Lübbe et al., 2006). DsrV, a homolog of precorrin-2 dehydrogenase, and DsrWa, a homolog of 235 uroporphyrin-III C-methyltransferase, may also be involved in siroheme biosynthesis 236 (Holkenbrink et al., 2011). DsrT is required for sulfide oxidation in Chlorobaculum tepidum, but 237 also found in SRM (Holkenbrink et al., 2011). The presence of dsrMK-paralogs (dsrM2, dsrK2) 238 upstream of dsrAB is not uncommon in SRM (Pereira et al., 2011). DsrMK are present in all 239 dsrAB-containing microorganisms and are a transmembrane module involved in reduction of 240 cytoplasmic DsrC-trisulfide in SRM, the final step in sulfate reduction (Santos et al., 2015). DsrC 241 encoded on the MAGs have the two essential cysteine residues at the C-terminal end for full 242 functionality (Venceslau et al., 2014). Interestingly, dsrC forms a gene duo with dsrL 243 downstream of dsrAB in all seven MAGs. This is surprising, because dsrL is not found in SRM but 244 in sulfur oxidizers. DsrL is highly expressed and essential for sulfur oxidation by the purple sulfur 245 bacterium Allochromatium vinosum (Lübbe et al., 2006; Weissgerber et al., 2014). DsrL is a 246 cytoplasmic iron-sulfur flavoprotein with proposed NAD(P)H:acceptor oxidoreductase activity 247 and was copurified with DsrAB from the soluble fraction of A. vinosum (Dahl et al., 2005). Given 248 the possible role of DsrL in sulfur oxidation, we sought to detect additional genes indicative of 249 oxidative sulfur metabolism in the acidobacterial MAGs. However, genes for sulfide:quinone 250 reductase (sqr), adenylyl-sulfate reductase membrane anchor subunit (aprM), flavocytochrome 251 c sulfide dehydrogenase (fccAB), sulfur reductase (sreABC), thiosulfate reductase (phsABC), 252 polysulfide reductase (psrABC), membrane-bound sulfite oxidizing enzyme (soeABC), 253 cytoplasmic sulfur trafficking enzymes (tusA, dsrE2, dsrEFH), or DsrQ/DsrU (unknown functions) 254 were absent (Laska et al., 2003; Holkenbrink et al., 2011; Lenk et al., 2012; Wasmund et al., 255 2017). SbA1, SbA3, SbA4, and SbA6 contain genes that have only low homology to 256 soxCD/sorAB, periplasmic sulfite-oxidizing enzymes (Supplementary Results) and, thus, might 257 have another function (Ghosh and Dam, 2009).

8 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 258 Despite ongoing sulfur cycling, concentrations of inorganic sulfur compounds such as sulfate are 259 low (lower µM range) in the Schlöppnerbrunnen II peatland (Schmalenberger et al., 2007; Küsel 260 et al., 2008; Knorr and Blodau, 2009). Enzymatic release of inorganic sulfur compounds from 261 organic matter might thus represent a significant resource for sulfur-dissimilating 262 microorganisms. We thus specifically searched for genes coding for known organosulfur 263 reactions that yield sulfite (Wasmund et al., 2017). Genes for cysteate sulfo-lyase (cuyA), 264 methanesulfonate monooxygenase (msmABCD), sulfoacetaldehyde acetyltransferase (xsc), and 265 taurine dioxygenase (tauD) were absent. However, suyAB, coding for the (R)-sulfolactate sulfo- 266 lyase complex that cleaves (R)-sulfolactate into pyruvate and sulfite (Denger and Cook, 2010), 267 were present in SbA4 and SbA5 (Supplementary Table S2a). Intriguingly, SbA4 and SbA5 only 268 have capability for sulfite reduction. SbA5 also encodes the racemase machinery for (S)- 269 sulfolactate to (R)-sulfolactate, (S)-sulfolactate dehydrogenase (slcC) and (R)-sulfolactate 270 dehydrogenase (comC); the regulator gene suyR or the putative importer SlcHFG were absent 271 (Denger and Cook, 2010). Pyruvate may be used as an energy and carbon source, while sulfite 272 could be used as an electron acceptor for anaerobic respiration (Simon and Kroneck, 2013).

273 Respiration 274 Cultivated Acidobacteria of subdivisions 1 and 3 are strict aerobes or facultative anaerobes 275 (e.g., Eichorst et al., 2007; Kulichevskaya et al., 2010, 2014; Pankratov and Dedysh, 2010; 276 Dedysh et al., 2012; Pankratov et al., 2012). Accordingly, we found respiratory chains encoded 277 in all acidobacterial MAGs (Figure 3, Supplementary Results), with (near) complete operons for 278 NADH dehydrogenase 1, succinate dehydrogenase (lacking in SbA2), one or both types of quinol 279 —cytochrome-c reductase, low-affinity terminal oxidases, and ATP synthase (lacking in SbA2) 280 (Supplementary Tables S2b–h). High-affinity terminal oxidases, putatively involved in 281 detoxification of oxygen (Ramel et al., 2013; Giuffrè et al., 2014), are limited to four MAGs 282 (Supplementary Table S2g). Genes for dissimilatory nitrogen or iron metabolisms are absent, 283 with the exception of a putative metal reductase in SbA2 of unclear physiological role 284 (Supplementary Results).

285 Hydrogen utilization and production 286 We identified [NiFe] hydrogenases of groups 1, 3, and 4 (Greening et al., 2016) in SbA1–7 287 (Supplementary Table S2j). Membrane-bound group 1 hydrogenases (SbA1, SbA3, SbA5) 288 consume hydrogen from the periplasm as an electron donor to generate energy, possibly 289 coupled to sulfate/sulfite reduction. In contrast to other Acidobacteria, no group 1h/5 290 hydrogenases, which are coupled to oxygen respiration, were identified (Greening et al., 2015). 291 Cytoplasmic group 3 hydrogenases (all MAGs) are bidirectional and proposed to be involved in 292 energy-generating hydrogen oxidation and/or fermentative hydrogen production. Membrane- 293 bound group 4 hydrogenases (SbA1, SbA5, SbA4, SbA6) produce H₂ and are postulated to

9 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 294 conserve energy by proton translocation by oxidizing substrates like formate (group 4a) or 295 carbon monoxide (via ferredoxin, group 4c) (Figure 3).

296 A versatile heterotrophic physiology 297 Acidobacteria are known for their capability to degrade simple and polymeric carbohydrates 298 (e.g., Kulichevskaya et al., 2010, 2014; Pankratov and Dedysh, 2010; Dedysh et al., 2012; 299 Eichorst et al., 2011; Pankratov et al., 2012; Rawat et al., 2012; Huber et al., 2016), supported 300 by many diverse carbohydrate-active enzymes encoded in their genomes (Ward et al., 2009; 301 Rawat et al., 2012). Accordingly, the MAGs recovered in our study also contain many genes 302 encoding diverse carbohydrate-active enzymes (Supplementary Methods, Figure 4). These 303 include glycoside hydrolases (GH, 1.0–4.0% of all genes), polysaccharide lyases (0.07–0.3%), 304 and carbohydrate esterases (0.7–1.4%) that are generally involved in degradation of complex 305 sugars, but also glycosyltransferases (0.9–1.4%) for biosynthesis of carbohydrates. Functional 306 GH gene groups (assigned by EC number) involved in cellulose and hemicellulose degradation 307 were most prevalent (Supplementary Table S4). Specifically, the most abundant EC numbers are 308 involved in cellulose (EC 3.2.1.4, e.g., GH5, GH74), xyloglucan (EC 3.2.1.150, EC 3.2.1.151, e.g., 309 GH5, GH74), or xylan (EC 3.2.1.8, EC 3.2.1.37, e.g., GH5) degradation, similar to other members 310 of Acidobacteria subdivision 1 and 3 (Ward et al., 2009; Rawat et al., 2012). Other abundant EC 311 numbers were associated with oligosaccharide degradation (EC 3.2.1.21, e.g., GH2) or α-N- 312 acetylgalactosaminidase genes (EC 3.2.1.49, e.g., GH109). Degradation of cellulose and 313 hemicellulose yields glucose and all MAGs encode glycolysis and pentose phosphate pathways 314 (Figure 3, Supplementary Results). α-N-acetylgalactosaminidase releases N-acetylgalactosamine 315 residues from glycoproteins that are commonly found in microbial cell walls and extracellular 316 polysaccharides (Bodé et al., 2013). N-acetylgalactosamine can not be directly utilized via 317 glycolysis, however the additionally required enzymes are present (Figure 3; Supplementary 318 Results). Under oxic conditions, organic carbon could be completely oxidized to CO₂ via the citric 319 acid cycle (Figure 3). Alternatively, we also identified fermentative pathways. SbA3 encodes the 320 bifunctional aldehyde-alcohol dehydrogenase AdhE that yields ethanol (Figure 3). All MAGs 321 encode additional aldehyde and alcohol dehydrogenases without clear substrate specificity that 322 could also ferment acetyl-CoA to ethanol. SbA7 and SbA5 encode a L-lactate dehydrogenase 323 (Ldh) yielding lactate from pyruvate, while six MAGs encode L-lactate dehydrogenases (LldD, 324 GlcDEF, LutABC) that presumably perform the reverse reaction (Figure 3). Similarly, we 325 identified pathways for acetate and/or propionate production or utilization in all MAGs (Figure 3; 326 Supplementary Results). SbA1 and SbA3 potentially produce H₂ via formate C-acetyltransferase 327 PflD, which cleaves pyruvate into acetyl-CoA and formate. SbA1 encodes for the membrane- 328 bound formate hydrogenlyase complex (fdhF, hyf operon) that produces H₂ and might also 329 translocate protons. SbA3 harbours an uncharacterized, cytoplasmic, monomeric FDH (fdhA) to 330 transform formate to H₂. SbA1, SbA3, SbA4, and SbA6 also encode membrane-bound,

10 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 331 periplasmic FDH (fdo operon) that transfers electrons into the membrane quinol pool, as a non- 332 fermentative alternative of formate oxidation (Figure 3, Supplementary Table S2j).

333 DsrAB-encoding Acidobacteria are metabolically active under anoxic conditions 334 We calculated the index of replication (iRep) with the native soil metagenomes to assess if the 335 DsrAB-encoding Acidobacteria were active in situ (Brown et al., 2016). SbA1 and SbA5, which 336 were sufficiently complete (≥75%) for accurate measurements, had iRep values of 1.21 and 337 1.19, respectively. This shows that a fraction of each population was metabolically active, i.e., 338 on average 21% of SbA1 and 19% of SbA5 cells were actively replicating at the time of 339 sampling. Concordantly, SbA1–7 were also transcriptionally active in the same native soil 340 samples. 35–46% of the SbA1–7 genes were expressed in at least one replicate. SbA1 and SbA5 341 contributed a considerable fraction (0.4% and 1.8%, respectively, Supplementary Table S1) of 342 the total mRNA reads in the native soil metatranscriptome. These data likely underestimate the 343 metabolic activity of SbA1–7 in situ because freshly sampled soil was stored at 4 °C for one 344 week prior to nucleic acids extraction.

345 We further analyzed metatranscriptome data from a series of anoxic incubations of the peat soil 346 with or without individual substrates (formate, acetate, propionate, lactate, or butyrate) and 347 with or without supplemental sulfate (Hausmann et al., 2016). While the incubations were not 348 designed to specifically test for the MAG-inferred metabolic properties, they still allowed us to 349 evaluate transcriptional response of the DsrAB-encoding Acidobacteria under various anoxic 350 conditions (Supplementary Methods and Results). All treatments triggered shifts in genome- 351 wide gene expression; more genes were significantly (p<0.05) upregulated (73–933) than 352 downregulated (14–81) as compared to the native soil. Upregulated genes included sulfur 353 metabolism, high-affinity terminal oxidases, group 1 and 3 hydrogenases, aldehyde-alcohol 354 dehydrogenase AdhE, glycoside hydrolases, and other carbon metabolism genes 355 (Supplementary Table S3). Significantly upregulated glycoside hydrolase genes were GH 2, 3, 5, 356 9, 10, 18, 20, 23, 26, 28, 29, 30, 33, 35, 36, 38, 43, 44, 50, 51, 55, 74, 76, 78, 79, 88, 95, 97, 357 105, 106, 109, and 129 genes in MAGs SbA1–6. None of the GH genes were significantly 358 downregulated in the incubations. Noteworthy genes that were significantly downregulated 359 were superoxide dismutases (sodA) in SbA2 and SbA4 (Supplementary Table S3a).

360 Discussion 361 Diverse members of the phylum Acidobacteria are abundant in various ecosystems, particularly 362 in soils and sediments with relative abundances typically ranging from 20–40% (Janssen, 2006). 363 Acidobacteria are currently classified in 26 subdivisions based on 16S rRNA phylogeny (Barns et 364 al., 2007). Given their phylogenetic breadth, comparably few isolates and genomes are 365 available to explore their metabolic capabilities. Yet strains in subdivisions 1, 3, 4, and 6 are

11 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 366 aerobic chemoorganotrophs that grow optimally at neutral or low pH (Dedysh, 2011; Eichorst et 367 al., 2011; Huber et al., 2014, 2016). Furthermore, subdivision 4 contains an anoxygenic 368 phototroph (Garcia Costas et al., 2012; Tank and Bryant, 2015), subdivisions 8 and 23 contain 369 anaerobes (Liesack et al., 1994; Coates et al., 1999; Losey et al., 2013), subdivisions 1, 3, and 370 23 fermentors (Pankratov et al., 2012; Kulichevskaya et al., 2014; Losey et al., 2013; Myers and 371 King, 2016) and subdivision 4, 8, 10 and 23 thermophiles (Izumi et al., 2012; Losey et al., 2013; 372 Crowe et al., 2014; Tank and Bryant, 2015).

373 Acidobacteria have previously been described as dominant inhabitants of wetlands worldwide, 374 namely members of subdivision 1, 3, 4, and 8 (Dedysh, 2011). Strains in the genera Granulicella 375 (Pankratov and Dedysh, 2010), Telmatobacter (Pankratov et al., 2012), Bryocella (Dedysh et al., 376 2012) and Bryobacter (Kulichevskaya et al., 2010) have been isolated from acidic wetlands and 377 are presumably active in plant-derived polymer degradation (such as cellulose) (Dedysh, 2011; 378 Pankratov et al., 2011; Schmidt et al., 2015; Juottonen et al., 2017), and in nitrogen and iron 379 cycling (Küsel et al., 2008; Kulichevskaya et al., 2014).

380 Here, we provide metagenomic and metatranscriptomic evidence that select Acidobacteria have 381 a dissimilatory sulfur metabolism. The seven acidobacterial MAGs from the Schlöppnerbrunnen 382 II peatland encode a complete dissimilatory sulfite or sulfate reduction pathway and represent 383 novel species of at least three novel genera in subdivision 1 or 3 (Supplementary Figure S3). 384 The phylogenetic separation into the two subdivisions in the concatenated marker gene tree is 385 also apparent in the DsrAB tree (Supplementary Figure S1). The acidobacterial DsrAB sequences 386 are distributed on two monophyletic clades within the uncultured family-level lineage 8 of the 387 reductive, bacterial-type DsrAB enzyme family branch (Müller et al., 2015). Furthermore, the 388 phylogenetic breadth of the acidobacterial DsrAB sequences is representative of the intra- 389 lineage sequence divergence, which suggests that the entire DsrAB lineage 8 represents yet 390 uncultivated bacteria of the phylum Acidobacteria. Members of this uncultured DsrAB lineage 391 are widespread in freshwater wetlands (Supplementary Figure S1) (Pester et al., 2012) and an 392 abundant fraction of the dsrAB diversity and permanent autochthonous inhabitants of oxic and 393 anoxic soil layers in the Schlöppnerbrunnen II peatland (Steger et al., 2011; Pester et al., 2010).

12 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 394 Presence of a complete gene set for canonical dissimilatory sulfate reduction suggests that the 395 pathway is functional, as the genetic capability for sulfate reduction can be rapidly lost by 396 adaptive evolution if unused (Hillesland et al., 2014). Except for a truncated aprB on SbA6, we 397 found no indications of pseudogenes, i.e., unexpected internal stop codons or reading frame 398 shifts, for any of the sulfate/sulfite reduction genes on the acidobacterial MAGs (Müller et al., 399 2015). In addition, sulfur genes of each MAG were expressed in the native soil and the anoxic 400 microcosms (Supplementary Table S3a). Many sulfur metabolism genes were also significantly 401 upregulated in the microcosms, with dsrC and aprBA among the top 10 most expressed genes in 402 SbA7 (Supplementary Table S3a). These findings further support full functionality of the 403 acidobacterial dissimilatory sulfur pathways under anoxic condition.

404 Known SRM typically couple sulfate respiration to oxidation of fermentation products such as 405 volatile fatty acids, alcohols, or hydrogen (Rabus et al., 2013). While other microorganisms in 406 the Schlöppnerbrunnen II soil, such as Desulfosporosinus, showed sulfate- and substrate-specific 407 responses in our microcosms, hundreds of acidobacterial 16S rRNA phylotypes did not (with the 408 exception of two) (Hausmann et al., 2016). Gene expression patterns of DsrAB-encoding 409 Acidobacteria in the individual anoxic microcosms as analyzed in the present study were 410 ambiguous. Genes for putative oxidation of the supplemented substrates (formate, acetate, 411 propionate, lactate, butyrate) were not specifically upregulated, neither with nor without 412 supplemental sulfate. However, sulfur metabolism genes were upregulated in some incubations 413 as compared to no-substrate-controls, suggesting indirect stimulation of sulfur dissimilation 414 (Supplementary Results, Supplementary Table S3a). Indirect changes in microbial activity after 415 the addition of fresh organic matter is often observed in soils (priming effects, Blagodatskaya 416 and Kuzyakov, 2008). One priming effect explanation is the co-metabolism theory stating that 417 easily available substrates provide the energy for microorganisms to produce extracellular 418 enzymes to make immobile carbon accessible, which is then also available to other 419 microorganisms. The DsrAB-encoding Acidobacteria have vast genetic capabilities for utilization 420 of carbohydrates and complete sugar degradation pathways (Figure 3), in accordance with 421 carbohydrate utilization potential previously described for subdivision 1 and 3 Acidobacteria 422 (Ward et al., 2009; Rawat et al., 2012). Yet carbohydrate utilization coupled to sulfate reduction 423 is a rare feature of known SRM [Cord-Ruwisch et al. (1986); Stetter (1988);]. While expression of 424 many of their glycoside hydrolase genes was upregulated in our anoxic peat soil microcosms, 425 further experiments are required to confirm that the DsrAB-encoding Acidobacteria couple 426 degradation of carbohydrate polymers or monomers to sulfate reduction.

427 It is intriguing to propose that MAGs SbA2, SbA3, and SbA7 derive from acidobacterial SRM as 428 they lack known sulfur oxidation genes, except dsrL, and express fully functional dissimilatory 429 sulfate reduction pathways (Supplementary Table S2a), including reductive, bacterial-type 430 dsrAB, and dsrD that may be exclusive to SRM (Hittel and Voordouw, 2000; Dahl and Friedrich,

13 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 431 2008; Ghosh and Dam, 2009; Rabus et al., 2015). An alternative hypothesis, however, is that 432 these novel Acidobacteria reverse the sulfate reduction pathway for dissimilatory sulfur 433 oxidation or sulfur disproportionation, as was recently shown for the deltaproteobacterium 434 Desulfurivibrio alkaliphilus (Thorup et al., 2017). D. alkaliphilus also lacks known sulfur oxidation 435 genes, except for sqr, and grows by coupling sulfide oxidation via an SRM-like pathway (with a 436 reductive-type DsrAB) to the dissimilatory reduction of nitrate/nitrite to ammonium. Sulfide 437 oxidation in acidobacterial MAGs SbA2, SbA3, and SbA7 could proceed analogous to the 438 pathway models by Thorup et al. (2017) and Christiane Dahl (Dahl, 2017). Briefly, hydrogen 439 sulfide might react with DsrC either spontaneously (Ijssennagger et al., 2015) or via an unknown 440 sulfur transfer mechanism to form persulfated DsrC. Persulfated DsrC is then oxidized by 441 DsrMKJOP, thereby transferring electrons into the membrane quinone pool, and releasing a 442 DsrC-trisulfide, which is the substrate for DsrAB (Santos et al., 2015; Dahl, 2017). It was 443 hypothesized that electrons released during DsrC-trisulfide oxidation to sulfite and DsrC are 444 transferred to DsrL (Dahl, 2017). Sulfite oxidation to sulfate would be catalyzed by AprBA- 445 QmoABC and Sat.

446 The acidobacterial MAGs have the genomic potential to use oxygen as terminal electron 447 acceptor and might thus couple sulfide oxidation to aerobic respiration. Alternative electron 448 acceptors for biological sulfur oxidation in wetlands could include nitrate/nitrite and metals such 449 as Fe(III) (Küsel et al., 2008). However, known genes for dissimilatory nitrate reduction and 450 metal reduction (Weber et al., 2006) were absent from these acidobacterial MAGs. Only SbA2 451 encodes a putative metal reduction complex that was recently characterized in 452 Desulfotomaculum reducens (Otwell et al., 2015). At this time, it is unclear whether DsrAB- 453 encoding Acidobacteria are capable of Fe(III) respiration, as seen in Geothrix fermentans 454 (Coates et al., 1999) and certain isolates in subdivision 1 (Blöthe et al., 2008; Kulichevskaya et 455 al., 2014).

456 Proposal of uncultivated acidobacterial genera 457 USulfotelmatobacter, USulfotelmatomonas, and USulfopaludibacter 458 Based on combined interpretation of phylogeny (concatenated phylogenetic marker genes, 459 DsrAB), genomic (ANI, AAI) and genetic (DsrAB) distances, and characteristic genomic features 460 of dissimilatory sulfur metabolism (Figure 3), in accordance with Konstantinidis et al. (2017), we 461 classify MAGs SbA1, SbA7, SbA5, SbA3, SbA4, and SbA6 into three new acidobacterial 462 uncultivated genera, including uncultivated species names for the >95% complete MAGs SbA1 463 and SbA5. In-depth phylogenomic analysis of SbA2 was not possible and therefore it is 464 tentatively assigned to Acidobacteria subdivision 3.

14 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 465 Acidobacteria subdivision 1

466  Uncultivated genus USulfotelmatobacter (Sul.fo.tel.ma.to.bac’ter. L. n. sulfur, sulfur; Gr. n. 467 telma, -tos, swamp, wetland; N.L. masc. n. bacter, bacterium; N.L. masc. n. 468 Sulfotelmatobacter, a bacterium with a dissimilatory sulfur metabolism from a swamp) 469 with USulfotelmatobacter kueseliae MAG SbA1 (kue.se’li.ae. N.L. gen. n. kueseliae, of 470 Kuesel, honouring Kirsten Küsel, for her work on the geomicrobiology of wetlands) and 471 USulfotelmatobacter sp. MAG SbA7.

472  USulfotelmatomonas gaucii MAG SbA5 (Sul.fo.tel.ma.to.mo.nas. L. n. sulfur, sulfur; Gr. n. 473 telma, -tos, swamp, wetland; N.L. masc. n. monas, unicellular organism; N.L. masc. n. 474 Sulfotelmatomonas, a bacterium with a dissimilatory sulfur metabolism from a swamp; 475 gau'.ci.i. N.L. gen. n. gaucii, of Gauci, in honour of Vincent Gauci, for his pioneering work 476 on the interplay of wetland sulfate reduction and global methane emission).

477 Acidobacteria subdivision 3

478  Uncultivated genus USulfopaludibacter (Sul.fo.pa.lu.di.bac’ter. L. n. sulfur, sulfur; L. n. 479 palus, -udis, L. swamp; N.L. masc. n. bacter, bacterium; N.L. masc. n. Sulfopaludibacter, a 480 bacterium with a dissimilatory sulfur metabolism from a swamp) with USulfopaludibacter 481 sp. MAG SbA3, USulfopaludibacter sp. MAG SbA4, and USulfopaludibacter sp. MAG SbA6.

482  Acidobacteria bacterium sp. MAG SbA2.

483 Conclusion 484 Sulfur cycling exerts important control on organic carbon degradation and greenhouse gas 485 production in wetlands, but knowledge about sulfur microorganisms in these globally important 486 ecosystems is scarce (Pester et al., 2012). Here, we show for the first time, using genome- 487 centric metagenomics and metatranscriptomics, that members of the phylum Acidobacteria 488 have a putative role in peatland sulfur cycling. The genomic repertoire of the novel 489 Acidobacteria species encompassed recognized acidobacterial physiologies, such as facultative 490 anaerobic metabolism, oxygen respiration, fermentation, carbohydrate degradation, and 491 hydrogen metabolism, but was additionally augmented with a DsrAB-based dissimilatory sulfur 492 metabolism (Figure 5). Integrating findings of sulfur oxidation in SRM and on reversibility of the 493 dissimilatory sulfate reduction pathway (Dannenberg et al., 1992; Fuseler and Cypionka, 1995; 494 Fuseler et al., 1996; Thorup et al., 2017) and co-occurrence of dsrD and dsrL, genes that are 495 considered characteristic for either sulfate reduction or sulfur oxidation (Dahl and Friedrich, 496 2008; Rabus et al., 2015), it is conceivable that the peatland Acidobacteria use the same 497 pathway for both sulfate reduction and sulfide oxidation. Some members that only encoded the

15 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 498 pathway for dissimilatory sulfite reduction had additional genes for sulfite-producing enzymes, 499 suggesting that organosulfonates may be a primary substrate for sulfur respiration. Our results 500 extend our understanding of the genetic versatility and distribution of dissimilatory sulfur 501 metabolism among recognized microbial phyla, but also underpin the challenge to 502 unambiguously differentiate between reductive or oxidative sulfur metabolism solely based on 503 (meta-)genome/transcriptome data (Thorup et al., 2017).

504 Conflict of Interest 505 The authors declare no conflict of interest.

506 Acknowledgements 507 We are grateful to Norbert Bittner for support during field sampling, Doris Steger and Pinsurang 508 Deevong for their contributions to qPCR analysis, and Florian Goldenberg for maintaining the 509 Life Science Computer Cluster at the Division of Computational Systems Biology (University of 510 Vienna). We thank the staff of the Joint Genome Institute (JGI) for metagenome and 511 metatranscriptome library preparation, sequencing, and standard bioinformatics support, 512 Bernhard Schink for help in naming of bacterial taxa, and Christiane Dahl, Petra Pjevac, Marc 513 Mußmann, and Kenneth Wasmund for valuable discussions and feedback. We acknowledge 514 LABGeM and the National Infrastructure France Genomique for providing the MicroScope MaGe 515 platform. The work conducted by the JGI was supported by the Office of Science of the U.S. 516 Department of Energy under Contract No. DE-AC02-05CH11231. This research was supported by 517 the Austrian Science Fund (FWF, P23117-B17, P25111-B22, P26392-B20, and I1628-B22), the JGI 518 (CSP 605), the German Research Foundation (DFG, PE 2147/1-1), and the European Union (FP7- 519 People-2013-CIG, Grant No PCIG14-GA-2013-630188).

520 References 521 Albertsen M, Hugenholtz P, Skarshewski A, Nielsen KL, Tyson GW, Nielsen PH. (2013). Genome 522 sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple 523 metagenomes. Nat Biotechnol 31: 533–538.

524 Barns SM, Cain EC, Sommerville L, Kuske CR. (2007). Acidobacteria phylum sequences in 525 uranium-contaminated subsurface sediments greatly expand the known diversity within the 526 phylum. Appl Environ Microbiol 73: 3113–3116.

527 Blagodatskaya Е, Kuzyakov Y. (2008). Mechanisms of real and apparent priming effects and their 528 dependence on soil microbial biomass and community structure: critical review. Biol Fertil Soils 529 45: 115–131.

16 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 530 Blöthe M, Akob DM, Kostka JE, Göschel K, Drake HL, Küsel K. (2008). pH gradient-induced 531 heterogeneity of Fe(III)-reducing microorganisms in coal mining-associated lake sediments. Appl 532 Environ Microbiol 74: 1019–1029.

533 Bodé S, Fancy R, Boeckx P. (2013). Stable isotope probing of amino sugars – a promising tool to 534 assess microbial interactions in soils. Rapid Commun Mass Spectrom 27: 1367–1379.

535 Brown CT, Olm MR, Thomas BC, Banfield JF. (2016). Measurement of bacterial replication rates in 536 microbial communities. Nat Biotechnol 34: 1256–1263.

537 Coates JD, Ellis DJ, Gaw CV, Lovley DR. (1999). Geothrix fermentans gen. nov., sp. nov., a novel 538 Fe(III)-reducing bacterium from a hydrocarbon-contaminated aquifer. Int J Syst Bacteriol 49: 539 1615–1622.

540 Cord-Ruwisch R, Ollivier B, Garcia J-L. (1986). Fructose degradation by Desulfovibrio sp. in pure 541 culture and in coculture with Methanospirillum hungatei. Curr Microbiol 13: 285–289.

542 Costello EK, Schmidt SK. (2006). Microbial diversity in alpine tundra wet meadow soil: novel 543 Chloroflexi from a cold, water-saturated environment. Environ Microbiol 8: 1471–1486.

544 Crowe MA, Power JF, Morgan XC, Dunfield PF, Lagutin K, Rijpstra IC et al. (2014). Pyrinomonas 545 methylaliphatogenes gen. nov., sp. nov., a novel group 4 thermophilic member of the phylum 546 Acidobacteria from geothermal soils. Int J Syst Evol Microbiol 64: 220–227.

547 Dahl C. (2017). Sulfur metabolism in phototrophic bacteria. In: Hallenbeck PC (ed). Modern 548 topics in the phototrophic prokaryotes. Springer, pp 27–66.

549 Dahl C, Engels S, Pott-Sperling AS, Schulte A, Sander J, Lübbe Y et al. (2005). Novel genes of the 550 dsr gene cluster and evidence for close interaction of Dsr proteins during sulfur oxidation in the 551 phototrophic sulfur bacterium Allochromatium vinosum. J Bacteriol 187: 1392–1404.

552 Dahl C, Friedrich CG. (2008). Microbial Sulfur Metabolism. Springer, Berlin Heidelberg.

553 Dannenberg S, Kroder M, Dilling W, Cypionka H. (1992). Oxidation of H₂, organic compounds and 554 inorganic sulfur compounds coupled to reduction of O₂ or nitrate by sulfate-reducing bacteria. 555 Arch Microbiol 158: 93–99.

556 Dedysh SN. (2011). Cultivating uncultured bacteria from northern wetlands: knowledge gained 557 and remaining gaps. Front Microbiol 2: 184.

558 Dedysh SN, Kulichevskaya IS, Serkebaeva YM, Mityaeva MA, Sorokin VV, Suzina NE et al. (2012). 559 Bryocella elongata gen. nov., sp. nov., a member of subdivision 1 of the Acidobacteria isolated

17 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 560 from a methanotrophic enrichment culture, and emended description of Edaphobacter 561 aggregans Koch et al. 2008. Int J Syst Evol Microbiol 62: 654–664.

562 Dedysh SN, Pankratov TA, Belova SE, Kulichevskaya IS, Liesack W. (2006). Phylogenetic analysis 563 and in situ identification of Bacteria community composition in an acidic Sphagnum peat bog. 564 Appl Environ Microbiol 72: 2110–2117.

565 Denger K, Cook AM. (2010). Racemase activity effected by two dehydrogenases in sulfolactate 566 degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase. 567 Microbiology 156: 967–974.

568 Eddy SR. (2011). Accelerated profile HMM searches. PLoS Comput Biol 7: e1002195.

569 Eichorst SA, Breznak JA, Schmidt TM. (2007). Isolation and characterization of soil bacteria that 570 define Terriglobus gen. nov., in the phylum Acidobacteria. Appl Environ Microbiol 73: 2708– 571 2717.

572 Eichorst SA, Kuske CR, Schmidt TM. (2011). Influence of plant polymers on the distribution and 573 cultivation of bacteria in the phylum Acidobacteria. Appl Environ Microbiol 77: 586–596.

574 Fuseler K, Cypionka H. (1995). Elemental sulfur as an intermediate of sulfide oxidation with 575 oxygen by Desulfobulbus propionicus. Arch Microbiol 164: 104–109.

576 Fuseler K, Krekeler D, Sydow U, Cypionka H. (1996). A common pathway of sulfide oxidation by 577 sulfate-reducing bacteria. FEMS Microbiol Lett 144: 129–134.

578 Garcia Costas AM, Liu Z, Tomsho LP, Schuster SC, Ward DM, Bryant DA. (2012). Complete 579 genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based 580 photoheterotroph belonging to the phylum Acidobacteria. Environ Microbiol 14: 177–190.

581 Ghosh W, Dam B. (2009). Biochemistry and molecular biology of lithotrophic sulfur oxidation by 582 taxonomically and ecologically diverse bacteria and archaea. FEMS Microbiol Rev 33: 999–1043.

583 Giuffrè A, Borisov VB, Arese M, Sarti P, Forte E. (2014). Cytochrome bd oxidase and bacterial 584 tolerance to oxidative and nitrosative stress. Biochim Biophys Acta 1837: 1178–1187.

585 Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB et al. (2016). Genomic and 586 metagenomic surveys of hydrogenase distribution indicate H₂ is a widely utilised energy source 587 for microbial growth and survival. ISME J 10: 761–777.

588 Greening C, Carere CR, Rushton-Green R, Harold LK, Hards K, Taylor MC et al. (2015). 589 Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging. Proc Natl Acad

591 Hausmann B, Knorr K-H, Schreck K, Tringe SG, Glavina del Rio T, Loy A et al. (2016). Consortia of 592 low-abundance bacteria drive sulfate reduction-dependent degradation of fermentation 593 products in peat soil microcosms. The ISME Journal 10: 2365–2375.

594 Hillesland KL, Lim S, Flowers JJ, Turkarslan S, Pinel N, Zane GM et al. (2014). Erosion of functional 595 independence early in the evolution of a microbial mutualism. Proc Natl Acad Sci USA 111: 596 14822–14827.

597 Hittel DS, Voordouw G. (2000). Overexpression, purification and immunodetection of DsrD from 598 Desulfovibrio vulgaris Hildenborough. Antonie Van Leeuwenhoek 77: 271–280.

599 Holkenbrink C, Barbas SO, Mellerup A, Otaki H, Frigaard N-U. (2011). Sulfur globule oxidation in 600 green sulfur bacteria is dependent on the dissimilatory sulfite reductase system. Microbiology 601 157: 1229–1239.

602 Huber KJ, Geppert AM, Wanner G, Fösel BU, Wüst PK, Overmann J. (2016). The first 603 representative of the globally widespread subdivision 6 Acidobacteria, Vicinamibacter silvestris 604 gen. nov., sp. nov., isolated from subtropical savannah soil. Int J Syst Evol Microbiol 66: 2971– 605 2979.

606 Huber KJ, Wüst PK, Rohde M, Overmann J, Foesel BU. (2014). Aridibacter famidurans gen. nov., 607 sp. nov. and Aridibacter kavangonensis sp. nov., two novel members of subdivision 4 of the 608 Acidobacteria isolated from semiarid savannah soil. Int J Syst Evol Microbiol 64: 1866–1875.

609 Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D, Walter MC et al. (2016). eggNOG 4.5: 610 a hierarchical orthology framework with improved functional annotations for eukaryotic, 611 prokaryotic and viral sequences. Nucleic Acids Res 44: D286–D293.

612 Hug La, Thomas BC, Sharon I, Brown CT, Sharma R, Hettich RL et al. (2016). Critical 613 biogeochemical functions in the subsurface are associated with bacteria from new phyla and 614 little studied lineages. Environ Microbiol 18: 159–173.

615 Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. (2010). Prodigal: prokaryotic 616 gene recognition and translation initiation site identification. BMC Bioinformatics 11: 119.

617 Ijssennagger N, Belzer C, Hooiveld GJ, Dekker J, van Mil SWC, Müller M et al. (2015). Gut 618 microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus 619 barrier in colon. Proc Natl Acad Sci USA 112: 10038–10043.

620 Ivanova AA, Wegner C-E, Kim Y, Liesack W, Dedysh SN. (2016). Identification of microbial

19 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 621 populations driving biopolymer degradation in acidic peatlands by metatranscriptomic analysis. 622 Mol Ecol 25: 4818–4835.

623 Izumi H, Nunoura T, Miyazaki M, Mino S, Toki T, Takai K et al. (2012). Thermotomaculum 624 hydrothermale gen. nov., sp. nov., a novel heterotrophic thermophile within the phylum 625 Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough. 626 Extremophiles 16: 245–253.

627 Janssen PH. (2006). Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S 628 rRNA genes. Appl Environ Microbiol 72: 1719–1728.

629 Juottonen H, Eiler A, Biasi C, Tuittila E-S, Yrjälä K, Fritze H. (2017). Distinct anaerobic bacterial 630 consumers of cellobiose-derived carbon in boreal fens with different CO₂/CH₄ production ratios. 631 Appl Environ Microbiol 83: e02533–16.

632 Kirschke S, Bousquet P, Ciais P, Saunois M, Canadell JG, Dlugokencky EJ et al. (2013). Three 633 decades of global methane sources and sinks. Nat Geosci 6: 813–823.

634 Knorr K-H, Blodau C. (2009). Impact of experimental drought and rewetting on redox 635 transformations and methanogenesis in mesocosms of a northern fen soil. Soil Biol Biochem 41: 636 1187–1198.

637 Konstantinidis KT, Rosselló-Móra R, Amann R. (2017). Uncultivated microbes in need of their own 638 taxonomy. ISME J. doi:https://doi.org/10.1038/ismej.2017.113[10.1038/ismej.2017.113].

639 Kraigher B, Stres B, Hacin J, Ausec L, Mahne I, van Elsas JD et al. (2006). Microbial activity and 640 community structure in two drained fen soils in the Ljubljana Marsh. Soil Biol Biochem 38: 2762– 641 2771.

642 Kulichevskaya IS, Suzina NE, Liesack W, Dedysh SN. (2010). Bryobacter aggregatus gen. nov., 643 sp. nov., a peat-inhabiting, aerobic chemo-organotroph from subdivision 3 of the Acidobacteria. 644 Int J Syst Evol Microbiol 60: 301–306.

645 Kulichevskaya IS, Suzina NE, Rijpstra WIC, Sinninghe Damsté JS, Dedysh SN. (2014). 646 Paludibaculum fermentans gen. nov., sp. nov., a facultative anaerobe capable of dissimilatory 647 iron reduction from subdivision 3 of the Acidobacteria. Int J Syst Evol Microbiol 64: 2857–2864.

648 Küsel K, Blöthe M, Schulz D, Reiche M, Drake HL. (2008). Microbial reduction of iron and 649 porewater biogeochemistry in acidic peatlands. Biogeosciences 5: 1537–1549.

650 Langmead B, Salzberg SL. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9: 651 357–359.

20 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 652 Laska S, Lottspeich F, Kletzin A. (2003). Membrane-bound hydrogenase and sulfur reductase of 653 the hyperthermophilic and acidophilic archaeon Acidianus ambivalens. Microbiology 149: 2357– 654 2371.

655 Lenk S, Moraru C, Hahnke S, Arnds J, Richter M, Kube M et al. (2012). Roseobacter clade bacteria 656 are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes. 657 ISME J 6: 2178–2187.

658 Liao Y, Smyth GK, Shi W. (2014). featureCounts: an efficient general purpose program for 659 assigning sequence reads to genomic features. Bioinformatics 30: 923–930.

660 Liesack W, Bak F, Kreft J-U, Stackebrandt E. (1994). Holophaga foetida gen. nov., sp. nov., a new, 661 homoacetogenic bacterium degrading methoxylated aromatic compounds. Arch Microbiol 162: 662 85–90.

663 Losey NA, Stevenson BS, Busse H-J, Sinninghe Damsté JS, Rijpstra WIC, Rudd S et al. (2013). 664 Thermoanaerobaculum aquaticum gen. nov., sp. nov., the first cultivated member of 665 Acidobacteria subdivision 23, isolated from a hot spring. Int J Syst Evol Microbiol 63: 4149–4157.

666 Love MI, Huber W, Anders S. (2014). Moderated estimation of fold change and dispersion for 667 RNA-seq data with DESeq2. Genome Biol 15: 550.

668 Loy A, Küsel K, Lehner A, Drake HL, Wagner M. (2004). Microarray and functional gene analyses 669 of sulfate-reducing prokaryotes in low-sulfate, acidic fens reveal cooccurrence of recognized 670 genera and novel lineages. Appl Environ Microbiol 70: 6998–7009.

671 Lübbe YJ, Youn H-S, Timkovich R, Dahl C. (2006). Siro(haem)amide in Allochromatium vinosum 672 and relevance of DsrL and DsrN, a homolog of cobyrinic acid a,c-diamide synthase, for sulphur 673 oxidation. FEMS Microbiol Lett 261: 194–202.

674 Meyer B, Imhoff JF, Kuever J. (2007). Molecular analysis of the distribution and phylogeny of the 675 soxB gene among sulfur-oxidizing bacteria: evolution of the Sox sulfur oxidation enzyme 676 system. Environ Microbiol 9: 2957–2977.

677 Müller AL, Kjeldsen KU, Rattei T, Pester M, Loy A. (2015). Phylogenetic and environmental 678 diversity of DsrAB-type dissimilatory (bi)sulfite reductases. ISME J 9: 1152–1165.

679 Myers MR, King GM. (2016). Isolation and characterization of Acidobacterium ailaaui sp. nov., a 680 novel member of Acidobacteria subdivision 1, from a geothermally heated Hawaiian microbial 681 mat. Int J Syst Evol Microbiol 66: 5328–5335.

682 Otwell AE, Sherwood RW, Zhang S, Nelson OD, Li Z, Lin H et al. (2015). Identification of proteins

21 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 683 capable of metal reduction from the proteome of the Gram-positive bacterium 684 Desulfotomaculum reducens MI-1 using an NADH-based activity assay. Environ Microbiol 17: 685 1977–1990.

686 Pankratov TA, Dedysh SN. (2010). Granulicella paludicola gen. nov., sp. nov., Granulicella 687 pectinivorans sp. nov., Granulicella aggregans sp. nov. and Granulicella rosea sp. nov., 688 acidophilic, polymer-degrading acidobacteria from Sphagnum peat bogs. Int J Syst Evol 689 Microbiol 60: 2951–2959.

690 Pankratov TA, Ivanova AO, Dedysh SN, Liesack W. (2011). Bacterial populations and 691 environmental factors controlling cellulose degradation in an acidic Sphagnum peat. Environ 692 Microbiol 13: 1800–1814.

693 Pankratov TA, Kirsanova LA, Kaparullina EN, Kevbrin VV, Dedysh SN. (2012). Telmatobacter 694 bradus gen. nov., sp. nov., a cellulolytic facultative anaerobe from subdivision 1 of the 695 Acidobacteria, and emended description of Acidobacterium capsulatum Kishimoto et al. 1991. 696 Int J Syst Evol Microbiol 62: 430–437.

697 Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. (2015). CheckM: assessing the 698 quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome 699 Res 25: 1043–1055.

700 Pereira IAC, Ramos AR, Grein F, Marques MC, Marques da Silva S, Venceslau SS. (2011). A 701 comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea. 702 Front Microbiol 2: 69.

703 Pester M, Bittner N, Deevong P, Wagner M, Loy A. (2010). A ’rare biosphere’ microorganism 704 contributes to sulfate reduction in a peatland. ISME J 4: 1591–1602.

705 Pester M, Knorr K-H, Friedrich MW, Wagner M, Loy A. (2012). Sulfate-reducing microorganisms in 706 wetlands – fameless actors in carbon cycling and climate change. Front Microbiol 3: 72.

707 Rabus R, Hansen TA, Widdel F. (2013). Dissimilatory sulfate- and sulfur-reducing prokaryotes. In: 708 Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds). The Prokaryotes – 709 prokaryotic physiology and biochemistry. Springer Berlin Heidelberg, pp 309–404.

710 Rabus R, Venceslau SS, Wöhlbrand L, Voordouw G, Wall JD, Pereira IAC. (2015). A post-genomic 711 view of the ecophysiology, catabolism and biotechnological relevance of sulphate-reducing 712 prokaryotes. Adv Microb Physiol 66: 55–321.

713 Ramel F, Amrani A, Pieulle L, Lamrabet O, Voordouw G, Seddiki N et al. (2013). Membrane-

22 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 714 bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris 715 Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation. 716 Microbiology 159: 2663–2673.

717 Rawat SR, Männistö MK, Bromberg Y, Häggblom MM. (2012). Comparative genomic and 718 physiological analysis provides insights into the role of Acidobacteria in organic carbon 719 utilization in Arctic tundra soils. FEMS Microbiol Ecol 82: 341–355.

720 Rodriguez-R LM, Konstantinidis KT. (2014). Bypassing cultivation to identify bacterial species. 721 Microbe Mag 9: 111–118.

722 Santos AA, Venceslau SS, Grein F, Leavitt WD, Dahl C, Johnston DT et al. (2015). A protein 723 trisulfide couples dissimilatory sulfate reduction to energy conservation. Science 350: 1541– 724 1545.

725 Saunois M, Bousquet P, Poulter B, Peregon A, Ciais P, Canadell JG et al. (2016). The global 726 methane budget 2000–2012. Earth Syst Sci Data 8: 697–751.

727 Sánchez-Andrea I, Rodríguez N, Amils R, Sanz JL. (2011). Microbial diversity in anaerobic 728 sediments at Río Tinto, a naturally acidic environment with a high heavy metal content. Appl 729 Environ Microbiol 77: 6085–6093.

730 Schmalenberger A, Drake HL, Küsel K. (2007). High unique diversity of sulfate-reducing 731 prokaryotes characterized in a depth gradient in an acidic fen. Environ Microbiol 9: 1317–1328.

732 Schmidt O, Horn MA, Kolb S, Drake HL. (2015). Temperature impacts differentially on the 733 methanogenic food web of cellulose-supplemented peatland soil. Environ Microbiol 17: 720– 734 734.

735 Serkebaeva YM, Kim Y, Liesack W, Dedysh SN. (2013). Pyrosequencing-based assessment of the 736 Bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on 737 poorly studied phyla and candidate divisions. PLoS One 8: e63994.

738 Simon J, Kroneck PMH. (2013). Microbial sulfite respiration.

739 Steger D, Wentrup C, Braunegger C, Deevong P, Hofer M, Richter A et al. (2011). 740 Microorganisms with novel dissimilatory (bi)sulfite reductase genes are widespread and part of 741 the core microbiota in low-sulfate peatlands. Appl Environ Microbiol 77: 1231–1242.

742 Stetter KO. (1988). Archaeoglobus fulgidus gen. nov., sp. nov.: a new taxon of extremely 743 thermophilic archaebacteria. Syst Appl Microbiol 10: 172–173.

23 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 744 Tank M, Bryant DA. (2015). Chloracidobacterium thermophilum gen. nov., sp. nov.: an 745 anoxygenic microaerophilic chlorophotoheterotrophic acidobacterium. Int J Syst Evol Microbiol 746 65: 1426–1430.

747 Thorup C, Schramm A, Findlay AJ, Finster KW, Schreiber L. (2017). Disguised as a sulfate 748 reducer: growth of the deltaproteobacterium Desulfurivibrio alkaliphilus by sulfide oxidation with 749 nitrate. MBio 8: e00671–17.

750 Tveit A, Schwacke R, Svenning MM, Urich T. (2013). Organic carbon transformations in high- 751 Arctic peat soils: key functions and microorganisms. ISME J 7: 299–311.

752 Urbanová Z, Bárta J. (2014). Microbial community composition and in silico predicted metabolic 753 potential reflect biogeochemical gradients between distinct peatland types. FEMS Microbiol Ecol 754 90: 633–646.

755 Vallenet D, Calteau A, Cruveiller S, Gachet M, Lajus A, Josso A et al. (2017). MicroScope in 2017: 756 an expanding and evolving integrated resource for community expertise of microbial genomes. 757 Nucleic Acids Res 45: D517–D528.

758 Venceslau SS, Stockdreher Y, Dahl C, Pereira IAC. (2014). The ‘bacterial heterodisulfide’ DsrC is 759 a key protein in dissimilatory sulfur metabolism. Biochim Biophys Acta 1837: 1148–1164.

760 Wang Y, Sheng H-F, He Y, Wu J-Y, Jiang Y-X, Tam NF-Y et al. (2012). Comparison of the levels of 761 bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of 762 illumina tags. Appl Environ Microbiol 78: 8264–8271.

763 Ward NL, Challacombe JF, Janssen PH, Henrissat B, Coutinho PM, Wu M et al. (2009). Three 764 genomes from the phylum Acidobacteria provide insight into the lifestyles of these 765 microorganisms in soils. Appl Environ Microbiol 75: 2046–2056.

766 Wasmund K, Mußmann M, Loy A. (2017). The life sulfuric: microbial ecology of sulfur cycling in 767 marine sediments. Environ Microbiol Rep. doi:https://doi.org/10.1111/1758- 768 2229.12538[10.1111/1758-2229.12538].

769 Watanabe T, Kojima H, Fukui M. (2016). Identity of major sulfur-cycle prokaryotes in freshwater 770 lake ecosystems revealed by a comprehensive phylogenetic study of the dissimilatory 771 adenylylsulfate reductase. Sci Rep 6: 36262.

772 Weber KA, Achenbach LA, Coates JD. (2006). Microorganisms pumping iron: anaerobic microbial 773 iron oxidation and reduction. Nat Rev Microbiol 4: 752–764.

774 Weissgerber T, Sylvester M, Kröninger L, Dahl C. (2014). A comparative quantitative proteomic

24 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 775 study identifies new proteins relevant for sulfur oxidation in the purple sulfur bacterium 776 Allochromatium vinosum. Appl Environ Microbiol 80: 2279–2292.

25 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

777 Figure legends

778 Figure 1 779 Microbial community composition in Schlöppnerbrunnen II peatland in samples from different 780 years and soil depths. (a) Abundance of phyla and proteobacterial classes in the native soil 781 (relative to all classified reads/amplicons). Taxa less abundant than 1% are grouped in grey. 782 Coverage abundance is based on metagenomic reads mapped to classified scaffolds. Amplicon 783 abundance is based on rrn operon-copy number-corrected abundance of 16S rRNA gene 784 operational taxonomic units (Hausmann et al., 2016). (b) Relative abundance of acidobacterial 785 subdivisions (SD) in the native soil samples as determined by 16S rRNA gene qPCR assays. In 786 addition, all subdivisions more abundant than 1% in a 16S rRNA gene amplicon dataset are 787 shown (Hausmann et al., 2016).

788 Figure 2 789 Organization of dissimilatory sulfur metabolism genes on acidobacterial MAGs SbA1–7. Red: sat; 790 orange: aprBA, qmoABC; green: dsrABCMKJOPM2K2; blue: dsrD; turquoise: dsrL; violet: 791 dsrNVWa; pink: suyAB, comC, slcC; white: genes of unknown function or not involved in sulfur 792 metabolism. In SbA2 all genes are on one scaffold (scaffold 0lkb). Gene fragments at contig 793 borders are indicated by an asterisk. aprB in SbA6, indicated by two asterisks, is truncated, 794 which indicates a pseudogene or is due to an assembly error. Scaffolds are separated by two 795 slashes.

796 Figure 3 797 Metabolic model as inferred from analysis of acidobacterial MAGs SbA1–7. Sulfur metabolism is 798 highlighted in yellow. Enzymes and transporters are shown in blue font. Glycoside hydrolases 799 are shown in pink font (Supplementary Table S2). Extracellular compounds are in parentheses. A 800 slash (/) indicates isozymes, i.e., enzymes that perform the same function, but are distinctly 801 different or have more than one established name. AcdA+B, MaeB+Pta, MeaB+Mce, Tal+Pgi: 802 bifunctional fusion genes/proteins. Otherwise the plus sign (+) indicates protein complexes. 803 TCA: tricarboxylic acid cycle. FDH: formate dehydrogenase. Hase: hydrogenase. NDH: NADH 804 dehydrogenase. HCO: haem-copper oxidase. TO: terminal oxidase. KDG: 2-dehydro-3-deoxy-D- 805 gluconate. KDGP: 2-dehydro-3-deoxy-D-gluconate 6-phosphate. Expression of at least one copy 806 of every enzyme and transporter was observed in the incubation samples.

26 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 807 Figure 4 808 Glycoside hydrolase genes are enriched in acidobacterial genomes/MAGs compared to genomes 809 from other taxa that encode DsrA/DsrB. DsrAB-containing MAGs SbA1–7 are shown as solid 810 symbols and numbered accordingly. X-axis shows the total number of predicted CDS per 811 genome/MAG.

812 Figure 5 813 Putative lifestyles of DsrAB-encoding Acidobacteria.

27 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 1.

a 2010 b 2004 2007 2010

100 60 SD 1 SD 2 SD 3 75 SD 13 40

Relative abundance (%) 25

0 0 0–5 cm 0–5 cm 0–5 5–10 cm 5–10 cm 5–10 Coverage 10–15 cm 10–15 cm 15–20 cm 10–15 cm 15–20 cm 20–30 cm 30–40 10–20 cm 10–20 cm 10–20 Amplicons 10–20 cm 10–20 cm 10–20 Amplicons Depth

Acidobacteria Actinobacteria Alphaproteobacteria

Betaproteobacteria Deltaproteobacteria Euryarchaeota

Firmicutes Gammaproteobacteria Planctomycetes

Thaumarchaeota Verrucomicrobia others bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 2.

dsrV dsrA dsrB* dsrL dsrC dsrM dsrK dsrO dsrP dsrWa dsrN SbA1 dsrT dsrJ dsrD sat qmoC qmoB qmoA aprA aprB dsrM2 dsrK2 dsrV dsrA dsrB dsrL dsrC dsrM dsrK dsrO dsrP* SbA7 dsrT dsrJ suyB comC slcC dsrM2 dsrK2 dsrV dsrA dsrB dsrL dsrC dsrM dsrK dsrO dsrP dsrWa dsrN SbA5 suyA dsrT dsrJ dsrD dsrD dsrN sat dsrM2 dsrK2 aprB aprA qmoA qmoB qmoC dsrA dsrB dsrL dsrC dsrM dsrK dsrO dsrP dsrV SbA2 dsrT dsrJ dsrD dsrN sat* dsrM2 dsrK2 aprB aprA* qmoA qmoB qmoCdsrA dsrB dsrL dsrC dsrM* dsrK dsrO dsrP SbA3 dsrT dsrJ suyB dsrN dsrM2 dsrK2 dsrA* dsrB dsrL dsrM dsrK* dsrOdsrP SbA4 5 kb suyA dsrD dsrC dsrJ dsrK2 dsrM2 dsrN dsrA dsrB dsrL dsrC dsrM dsrK dsrO dsrP SbA6 dsrD aprB** dsrT dsrJ bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Fig. 3.

+ [2 H ] [H2] [H+] [H+] [H+] [NiFe] hydrogenases group 4a group 4c group 4f group 1 HyfD–I CooM–H EhfA–F e– + FdhF

+ + + + + + + + H H 2 H + NADPH H2 + NADP H2 + Fdox 2 H + Fdred H2 + NAD 2 H + NADH H 2 H⁺ H₂ + HyhBCSL HdrA / HdrL + MvhDCA HoxEFUYH + Xred Xox + H2 formate + H CO2 + H2 group 3b group 3c group 3d + Fdred Fdox

FDH GalNAc Pentose phosphate Entner–Doudoroff [formate] Glk / GlcK Glycolysis FdoGHI degradation pathway pathway [CO + H+] Zwf 2 N-acetylgalactosamine-R glucose glucose 6-phosphate 6-phosphogluconolactone KDG

e– GH109 Pgi / Tal+Pgi Pgl KdgK Edd N-acetylgalactosamine + R fructose 6-phosphate 6-phosphogluconate KDGP (hemi-)cellulose GlcK (?) PfkA / Pfp / PfkB Gnd / YqeC KdgA e.g., GH5, N-acetylgalactosamine 6-phosphate fructose 1,6-bisphosphate ribulose 5-phosphate GH74 + ribulose glyceraldehyde pyruvate NagA FbaA / FbaB disaccharides 5-phosphate 3-phosphate e.g., galactosamine 6-phosphate Rpe RpiA / RpiB GH2 TpiA Sulfur metabolism AgaS dihydroxyacetone glyceraldehyde xylulose 5-phosphate ribose 5-phosphate phosphate 3-phosphate glucose, tagatofuranose 6-phosphate PPi galactose, … GapA + + Tkt / TktCD H HppA [H ] PfkB 1,3-bisphosphoglycerate Pi glyceraldehyde 3-phosphate + tagatofuranose 1,6-bisphosphate Pgk sedoheptulose 7-phosphate LacD Tal / Tal+Pgi 3-phosphoglycerate sulfate [sulfate] ATP NDH-1 dihydroxyacetone glyceraldehyde GpmA / GpmI SulP or DASS erythrose 4-phosphate fructose (complex I) phosphate 3-phosphate 2-phosphoglycerate Ppa Tkt / TktCD + xylulose 6-phosphate 2 Pi PPi Sat NADH Eno (again) 5-phosphate [H+] NuoA–N H+ Ppc APS NAD+ PEP glyceraldehyde fructose AprBA Pyk e– PckA 3-phosphate 6-phosphate Adk QmoABC pyruvate 2 ADP AMP e– MaeA / MaeB+Pta NDH-2 ATP sulfite e– (complex I) PfoA / PorCDAB (Fd) DsrCred PflD e– NADH ? DsrAB Ndh DsrL (?) + NAD AceEF + Lpd (NAD) FdhA formate CO₂ DsrC-trisulfite e– FumA/FumB / DsrMKJOP FumC malate oxaloacetate GltA / CitZ / CitA / CitB / CitY AdhE AdhE – Mdh acetyl-CoA acetaldehyde ethanol sulfitepyruvate e

Pta / sulﬁde SDH citrate TCA fumarate MaeB+Pta SuyAB (complex II) AcdA+B / YdiF Acs ⁻ (2R)-3-sulfolactate SdhC(D)AB AcnA acetyl-P e transport NAD⁺ NADH [sulﬁde] Kdg, SsdA ComC e– succinate isocitrate AckA 3-sulfopyruvate DsrD DsrN SucCD Icd FlxDCBA acetate Fdox Xox SlcC DsrV DsrWa succinyl-CoA 2-oxoglutarate HdrCBA SucAB + Lpd Fd X (2S)-3-sulfolactate DsrT DsrM2K2 SbmAB / red red MutAB ScpB ScpC / YdiF RR-CoA Mce / MeaB+Mce PccBAC (R)-methylmalonyl-CoA (S)-methylmalonyl-CoA propionyl-CoA propionate [propionate]

Propionate metabolism Acs Oxidative stress Lactate metabolism superoxide LldD / GlcDEF / LutABC succinate PrpB AcnA PrpD PrpC (2R,3S)-2-methylisocitrate cis-2-methylaconitate (2S,3S)-2-methylcitrate propionyl-CoA SodA + pyruvate SodC H₂O₂ + O₂ glutathione Ared pyruvate L-lactate [L-lactate] SD1 SbA1 SbA7 SbA5 Gpo KatG LutP SbA2 SbA3 SbA4 SbA6 SD3 glutathione O₂ Aox Ldh disulﬁde all gene(s) present gene(s) absent most ? All genes are expressed + H+ H+ H EtfBA + e– O2 H2O O2 H2O O2 + H H2O ADPATP pyruvateacetate H+ H+

PetCB(D) ActA–G Cta/Cox CcoNO CydAB AtpA–G PoxB EqoA e– e– e– e– e– e– e– e– e–

[H+] [H+] [H+] [H+] [H+] [H+] bc₁ alternative HCO class A HCO class C bd-type ATP synthase complex III complex III complex IV complex IV complex IV (complex V) low-affinity TO high-affinity TO high-affinity TO bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 4.

Acidobacteria genomes DsrAB-encoding genomes A5 4

A2 3 A3

A1 A4 Caldivirga 2

Gemmatimonas Thermoproteus 1 A7 Fraction of genes with predicted GH domains (%)

0 3 6 9 12 0 3 6 9 12 Number of predicted CDS (× 1000)

Acidobacteria Actinobacteria Alphaproteobacteria

Betaproteobacteria Chlorobi Crenarchaeota

Deltaproteobacteria Euryarchaeota Firmicutes

Gammaproteobacteria Gemmatimonadetes Nitrospirae

Thermodesulfobacteria bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 5.

Anaerobic CO₂ + H₂ Aerobic

acetate Carbo- hydrates O₂ Carbo- e⁻ Fermentation ethanol hydrates Aerobic respiration CO₂ H₂O lactate

CO₂ propionate H₂O Sulfate/sulﬁte reduction H₂S H₂S Carbohydrates Sulfur oxidation O₂ e⁻ e⁻

H₂ SO₄²⁻ ?ox SO₄²⁻ SO₃²⁻ Organic S H⁺ ?red bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Peatland Acidobacteria with a dissimilatory sulfur 2 metabolism: Supplementary Information

3 Supplementary Methods

4 Quantitative PCR analysis of acidobacterial subdivisions 5 Acidobacteria subdivisions 1, 2, and 3 were separately quantified using 16S rRNA gene-targeted 6 real-time quantitative PCR (qPCR) assays. Coverage of the subdivisions was estimated using the 7 RDP ProbeMatch online tool with RDP Release 11, Update 5, good quality filter applied, requiring 8 a full match to the probe sequence (Cole et al., 2014). The following parameters ensure 9 optimized efficiency and sensitivity of each qPCR assay. Subdivision 1: Acid303Fa/Acid303Fb (5′- 10 GCG CAC GGM CAC ACT GGA-3′/5′-GCG CGC GGC CAC ACT GGA-3′) and Acid657R (5′-ATT CCA 11 CKC ACC TCT CCC AY-3′), 76%/0.1% coverage by primers pairs, primer concentration: 1000 nM, 12 annealing temperature: 68.5 °C; Subdivision 2: Acid702Fa/Acid702Fb (5′-AGA TAT CTG CAG GAA 13 CAY CC-3′/5′-AGA TAT CCG CAG GAA CAT CC-3′) and Acid805R (5′-CTG ATS GTT TAG GGC TAG-3′), 14 64%/7% coverage, primer concentration: 1000 nM, annealing temperature: 62.5 °C; Subdivision 15 3: Acid306F (5′-CAC GGC CAC ACT GGC AC-3′) and Acid493R (5′-AGT TAG CCG CAG CTK CTT CT- 16 3′), 77% coverage, primer concentration: 500 nM, annealing temperature: 69 °C. Thermal 17 cycling was carried out with an initial denaturation (94 °C) followed by 40–45 cycles of 18 denaturation (94 °C, 40 s), annealing (68.5 °C, 62.5 °C, or 69 °C, 40 s), and elongation (72 °C, 19 40–45 s). PCR efficiency with perfectly-matched reference targets was between 82–86% with an 20 R² of 0.99 and a limit of detection at 100 target genes per reaction. For calculation of relative 21 abundances, total bacterial and archeal 16S rRNA genes were quantified using a previously 22 published qPCR assay (Pester et al., 2010; Hausmann et al., 2016).

23 Metagenomic and metatranscriptomic sequencing 24 DNA was sent to the JGI, where it was fragmented to a target length of 270 nt. Libraries were 25 generated with the KAPA-Illumina library creation kit (KAPA biosystems) and sequenced on an 26 Illumina HiSeq2000 sequencer. The native soil yielded 232 million 150 nt paired-end reads. 27 Three sequencing libraries of the pooled DNA-SIP sample yielded 273, 52, and 350 million 28 150 nt paired-end reads each. DNA from the native soil was also sent to the King Abdullah 29 University of Science and Technology (Thuwal, Saudi Arabia), libraries prepared with the Nextera 30 DNA Library Prep kit (Illumina), and sequenced on an Illumina HiSeq2000 sequencer (179 million 31 101 nt paired-end reads).

32 Triplicate RNA samples from the native soil and from each incubation treatment and time point 33 were sent to the JGI. When possible, rRNA was depleted using Ribo-Zero rRNA removal kit

1 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 34 (Epicentre). cDNA libraries were generated with the Truseq Stranded RNA LT kit (Illumina) and 35 sequencing was performed on an Illumina HiSeq2000 sequencer. One propionate- and sulfate- 36 stimulated replicate microcosm was excluded because of inconsistent response in sulfate 37 turnover as compared to the other two replicates (Hausmann et al., 2016), resulting in a total of 38 73 samples with 27–188 million 150 nt paired-end reads.

39 Gene- and genome-based taxonomic classification and phylogeny 40 Phylogenetic reconstruction of DsrAB sequences was performed based on an established DsrAB 41 alignment (Müller et al., 2015). DsrAB amino acid sequences from the MAGs and unbinned 42 scaffolds were aligned to the filtered seed alignment using MAFFT 7.271 (Katoh and Standley, 43 2013). De novo maximum likelihood trees were calculated with FastTree 2.1.9 using the LG 44 model and 1000 resamplings (Price et al., 2010). Phylogenetic distances of the DsrAB 45 sequences of the MAGs and scaffolds to other DsrAB harbouring organisms were calculated with 46 T-Coffee 11 (Notredame et al., 2000) using the unfiltered reference alignment without the 47 intergenic region (Müller et al., 2015). Trees were visualized with iTOL (Letunic and Bork, 2016) 48 and Inkscape (inkscape.org).

49 Representative genome assemblies from the phylum Acidobacteria and outgroups from the 50 Proteobacteria, Firmicutes, and Verrucomicrobia were obtained from NCBI for phylogenomic 51 analysis. A filtered and concatenated amino acid alignment of 34 phylogenetically informative 52 marker genes was created using CheckM (Parks et al., 2015). Phylobayes was used to calculate 53 the tree with a CAT-GTR model (Lartillot et al., 2009). Phylobayes was run in five independent 54 chains for 11000 cycles each (corresponding to approx. 6.8×10⁶ tree generations per chain). 55 The first 6000 cycles in each chain were discarded as burn in (corresponding to approx. 3.7×10⁶ 56 tree generations per chain).

57 Pairwise average nucleic and amino acid identities (ANI, AAI) between all protein-coding genes 58 of each MAG and published reference genomes were calculated to estimate novelty (adapted 59 from Varghese et al., 2015). Two-way ANI and AAI were calculated based on reciprocal best blast 60 hits filtered for sequence identity (≥70% and ≥30% for ANI and AAI, respectively) and 61 alignment length (≥70% of the shorter sequence). Average identities and alignment fractions 62 (AF) for each comparisons were calculated as outlined previously (Varghese et al., 2015). None 63 of the comparisons reached an ANI above the intra-species threshold of 96.5% (Varghese et al., 64 2015). Due to lack of a generic intra-genus AAI threshold, we used the existing acidobacterial 65 taxonomy as a reference. Intra-genus AAI variability of published acidobacterial genera with 66 more than one species (Acidobacterium, Granulicella, Terriglobus) ranged from 60–71% 67 (alignment fraction 52–66%).

2 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 68 Manually curated annotation of of DsrAB-encoding genomes 69 All genes of interest were manually curated using MaGe (Vallenet et al., 2017). This included 70 assessment of best-BLAST-hits to reference genomes and UniProt entries (The UniProt 71 Consortium, 2015), presence of the required functional domains (InterPro/InterProScan; Mitchell 72 et al., 2015; Jones et al., 2014) and, if appropriate, transmembrane helices (TMhmm; Krogh et 73 al., 2001), membership in the correct COGs, and membership of syntenic regions (operons). 74 COGs in MaGe are assigned using COGnitor (www.ncbi.nlm.nih.gov/COG/) which are often too 75 broad to be of use, therefore we additionally classified all coding DNA sequences (CDS) using 76 the bactNOG database (eggNOG, Huerta-Cepas et al., 2016). All possible HMM profiles were 77 matched against every gene (E-value threshold 1) and only the best hit was extracted. This non- 78 stringent E-value threshold allowed very small genes and fragments to be classified as well.

79 Carbohydrate-active enzymes 80 Carbohydrate-active enzymes (Lombard et al., 2014, www.cazy.org) in the MAGs were identified 81 and classified with dbCAN 4.0 (Yin et al., 2012). For comparison, genomes belonging to the 82 Acidobacteria and to genera with DsrAB-encoding members were downloaded from NCBI (June 83 2017) and analyzed with dbCAN as well. Genera with DsrAB-encoding members were identified 84 based on literature research and UniProt InterPro/TIGRFAM searches for DsrA 85 (IPR011806/TIGR02064) and DsrB (IPR011808/TIGR02066). For comparability and consistency, 86 de novo ORF predictions were performed with prodigal (Hyatt et al., 2010) for all genomes and 87 MAGs. Presence of DsrA and/or DsrB was again verified with the TIGRFAM models and HMMER. 88 dbCAN’s HMM profiles were identified with HMMER and parsed with the provided dbCAN script 89 and R (R Core Team, 2017). Analysed acidobacterial genomes belonged to the genera 90 Acidobacterium, Bryobacter, Chloracidobacterium, Edaphobacter, Geothrix, Granulicella, 91 Holophaga, Koribacter, Luteitalea, Pyrinomonas, Silvibacterium, Solibacter, Terracidiphilus, 92 Terriglobus, and Thermoanaerobaculum. 82 additional acidobacterial genomes without genus 93 classification were also analysed. DsrA/DsrB-encoding genomes derived from the genera 94 Acetonema, Achromatium, Acidiferrobacter, Alkalilimnicola, Allochromatium, Ammonifex, 95 Anaeromyxobacter, Archaeoglobus, Azospirillum, Bilophila, Caldimicrobium, Caldivirga, 96 Carboxydothermus, Chlorobaculum, Chlorobium, Curvibacter, Desulfacinum, Desulfamplus, 97 Desulfarculus, Desulfatibacillum, Desulfatiglans, Desulfatirhabdium, Desulfatitalea, 98 Desulfitibacter, Desulfitobacterium, Desulfobacca, Desulfobacter, Desulfobacterium, 99 Desulfobacula, Desulfobulbus, Desulfocapsa, Desulfocarbo, Desulfococcus, Desulfocurvus, 100 Desulfofervidus, Desulfofustis, Desulfohalobium, Desulfoluna, Desulfomicrobium, Desulfomonile, 101 Desulfonatronospira, Desulfonatronovibrio, Desulfonatronum, Desulfonauticus, Desulfonispora, 102 Desulfopertinax, Desulfopila, Desulfoplanes, Desulforegula, Desulforhopalus, Desulforudis, 103 Desulfosarcina, Desulfospira, Desulfosporosinus, Desulfotalea, Desulfothermus, Desulfotignum, 104 Desulfotomaculum, Desulfovermiculus, Desulfovibrio, Desulfovirgula, Desulfurella,

3 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 105 Desulfurispora, Desulfurivibrio, Dethiosulfatarculus, Dissulfuribacter, Ferriphaselus, Gallionella, 106 Gemmatimonas, Gordonibacter, Gracilibacter, Halodesulfovibrio, Halorhodospira, Lamprocystis, 107 Lautropia, Magnetococcus, Magnetomorum, Magnetoovum, Magnetospira, Magnetospirillum, 108 Magnetovibrio, Marichromatium, Moorella, Pelodictyon, Phaeospirillum, Prosthecochloris, 109 Pyrobaculum, Rhodomicrobium, Rubrivivax, Ruegeria, Ruthia, Sedimenticola, Sideroxydans, 110 Sulfuricella, Sulfuritalea, Syntrophobacter, Syntrophomonas, Syntrophus, Thermanaeromonas, 111 Thermocladium, Thermodesulfatator, Thermodesulfobacterium, Thermodesulfobium, 112 Thermodesulforhabdus, Thermodesulfovibrio, Thermoproteus, Thermosinus, 113 Thermosulfurimonas, Thioalkalivibrio, Thiobacillus, Thiocapsa, Thiocystis, Thiodiazotropha, 114 Thioflavicoccus, Thioflexothrix, Thioglobus, Thiohalocapsa, Thiohalomonas, Thiolapillus, 115 Thiomargarita, Thioploca, Thiorhodococcus, Thiorhodovibrio, Thiosymbion, Thiothrix, and 116 Vulcanisaeta.

117 Expression analysis 118 Metatranscriptomic reads were quality filtered at the JGI using their analysis pipeline. In short, 119 the raw reads were quality-trimmed to Q10, adapter-trimmed using bbduk (minimal allowed 120 length 50 nt), followed by removal of PhiX control sequences, artefacts, human sequences, and 121 reads containing N bases with bbduk/bbmap (BBTools, http://jgi.doe.gov/data-and- 122 tools/bbtools/). rRNA reads were removed using the SILVA database (Quast et al., 2013) and 123 bbmap. This resulted in 73 samples with 22–161 million high quality non-rRNA reads with a 124 median length of 150 nt. Those were then mapped to the combined metagenomic assembly 125 using bowtie2 with the default scoring function (Langmead and Salzberg, 2012). Fragments per 126 CDS were then independently counted using featureCounts 1.5.0 (Liao et al., 2014). Differential 127 expression analysis of SbA1–7 was performed using R (R Core Team, 2017) and the DESeq2 128 package (Love et al., 2014).

129 Supplementary Results and Discussion

130 Sulfite dehydrogenase homologs 131 We identified several genes encoding putative sulfite dehydrogenases of the COG2041 family: 132 (A) SbA3 and SbA4 encode orthologs to Cupriavidus necator (Ralstonia eutropha) N-1 soxC 133 (CNE_1c35220), Sbm_v1_d1920025 and Sbm_v1_e3730002, respectively (~50% sequence 134 identity). Directly downstream are genes homologous to the N-terminal region of C. necator 135 soxD (CNE_1c35210), Sbm_v1_d1920026 and Sbm_v1_e3730003, respectively (~40% sequence 136 identity at <50% overlap). soxABXYZ, present in C. necator, are not found on any acidobacterial 137 MAG. However, C. necator N-1 can not oxidize thiosulfate (Dahl and Friedrich, 2008). (B) Ca. 138 Solibacter usitatus encodes a sorAB-like gene pair (Acid_7248–7249), which we also found in 139 SbA1 (Sbm_v1_b530003–4), SbA3 (Sbm_v1_d50016–17), SbA4 (Sbm_v1_e5130014–13 and

150 Respiration and oxidative stress 151 Respiration with oxygen as terminal electron acceptor requires a membrane electron transport 152 chain involving up to four complexes, i.e., the NADH dehydrogenase (NDH, respiratory complex 153 I), the succinate dehydrogenase (SDH, respiratory complex II), the quinol—cytochrome-c 154 reductase (cytochrome bc₁ complex or alternative complex III, respiratory complex III), and the 155 terminal oxidase (cytochrome-c oxidase or cytochrome bd-type oxidase, respiratory complex 156 IV). Complexes I (NDH-1 only), III, and IV (except bd-type) translocate protons through the cell 157 membrane building up proton motive force. The ATP synthase uses the proton motive force to 158 generate ATP and is called respiratory complex V. SbA5 and SbA7 encode every gene for 159 complexes I–V, while SbA1, SbA3, SbA4, and SbA6 encode only partial operons for some 160 complexes. SbA2, the most incomplete MAG, is lacking all genes of complex II and V (Figure 3).

161 Oxidation of organic matter generates reducing equivalents, e.g., in glycolysis one NADH is 162 formed per one glucose. NADH is oxidized to NAD⁺/H⁺ by the NDH, which in turn transfers 163 electron to the membrane quinone pool. Two types of NDH are characterized in E. coli. NDH-1, 164 consisting of a large complex encoded by the nuo operon, translocates protons, while NDH-2, 165 encoded by a single gene (ndh), does not. We identified both NDH-1 and NDH-2 in the MAGs, 166 with the latter (partially) missing in SbA2, SbA4, and SbA7. All MAGs harbour one or more 167 (partially fragmented) nuoACDHJKLMN operons. One operon in each MAG also includes nuoEFG 168 (except in SbA3). NuoEFG forms the catalytic NADH dehydrogenase module of complex I, while 169 NuoBCDHIN and NuoKLM form the hydrogenase and transporter modules, respectively. NuoA 170 and NuoJ are not part of the modules and likely involved in assembly of the complex (Friedrich 171 et al., 2016). SbA6 is missing nuoI, likely because of MAG incompleteness (Supplementary Table 172 S2b).

173 SDH is encoded by the sdh operon. It consists of a cytoplasmic-facing catalytic subunit (SdhAB) 174 and a transmembrane cytochrome b₅₅₆ or b₅₅₈ subunit (SdhCD in E. coli or larger SdhC in B. 175 subtilis), which together transfer the electrons from oxidation of succinate to fumarate into the

5 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 176 membrane quinone pool. It is the only complex of the respiration chain not involved in proton 177 translocation. Both complex I and II are found in many anaerobically respiring microorganisms, 178 including SRM (e.g., Pereira et al., 2011; Klenk et al., 1997; Rabus et al., 2004; Strittmatter et 179 al., 2009; Plugge et al., 2012; Visser et al., 2013; Kuever et al., 2014; Mardanov et al., 2016). We 180 identified SDH in all MAGs but SbA2, arranged like the B. subtilis-type operon (sdhCAB) 181 (Supplementary Table S2c). SbA1 harbours a second operon (sdhACDB) that could also be a 182 fumarate reductase (frdACDB). Fumarate reductase performs the reverse reaction of SDH as 183 part of the reductive citric acid cycle, but both enzymes were shown to catalyze both reaction in 184 E. coli (Guest, 1981; Maklashina et al., 1998). No other MAG harboured candidates for fumarate 185 reductase.

186 Complex III, the quinol—cytochrome-c reductase transfers electron from the membrane quinone 187 pool to cytochrome c. Cytochrome c is then oxidized and O₂ is reduced to H₂O by a cytochrome- 188 c terminal oxidase. Alternately, a quinol terminal oxidase can directly utilize electrons from the 189 membrane quinone pool to reduce O₂. Two isofunctional complexes of quinol—cytochrome-c 190 reductases are known. The two component cytochrome bc₁ complex is encoded by the pet 191 operon and present in all MAGs but SbA2 (Supplementary Table S2d). Alternative complex III 192 (ACIII) (Refojo et al., 2012) with seven subunits is encoded by the act operon and present in all 193 MAGs but SbA7 (Supplementary Table S2e). Some of the terminal oxidase genes are found 194 immediately down- or upstream of quinol—cytochrome-c reductase operons (Supplementary 195 Tables S2e–g), as is observed in other Acidobacteria (e.g., Ca. K. versatilis, Ca. S. usitatus, 196 Chloracidobacterium thermophilum; Garcia Costas et al., 2012). In between alternative complex 197 III and the terminal oxidase genes, we always find the sco gene coding for a chaperone of the 198 SCO1/SenC family, a putative assembly factor for respiratory complexes (Buggy and Bauer, 199 1995). We identified both families of terminal oxidases, i.e., haem-copper oxidases (HCO) of 200 classes A and C, which are cytochrome-c oxidases, and cytochrome bd-type oxidases, which are 201 quinol oxidases. HCO class A are classified as low-affinity terminal oxidases (LATO) in contrast to 202 HCO class C and bd-type oxidases that are high-affinity terminal oxidases (HATO) (Morris and 203 Schmidt, 2013). Three distinct operon structures for HCO family A were observed in SbA1–7 and 204 also other Acidobacteria, e.g. Ca. K. versatilis and Ca. S. usitatus: (1) downstream of ACIII and 205 sco, (2) upstream of petBC (not found in Ca. K. versatilis), both named ctaCDEF, and (3) without 206 quinol—cytochrome-c reductase genes found up- or downstream but with a subunit III consisting

207 of two genes (coxOP) (Supplementary Table S2f). The subunit II of all three types have the CuA 208 copper center motifs (IPR001505), which is found in cytochrome-c oxidases but not in quinol 209 oxidases (Pereira et al., 2001). The essential subunits are I and II (Pereira et al., 2001) and are 210 present in all MAGs but SbA4. High-affinity terminal oxidases of HCO class C or bd-type oxidases 211 are present in SbA1, SbA5, SbA3, and SbA6 (Supplementary Table S2g). Both types are encoded 212 by two subunits on the MAGs. Secondary genes, as found in other organisms, e.g., ccoQP 213 (Bühler et al., 2010) or cydS/cydX (Cook and Poole, 2016), are missing.

6 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 214 An ATP synthase of the FₒF₁-ATPase type is present in all MAGs but SbA2 (Supplementary Table 215 S2h). Its genes are consistently split into two operons, atpZIBE and atpF′FHAGDC. The former is 216 missing in SbA6, while the later is fragmented in SbA1 with atpG missing completely. atpF and 217 atpF′ are paralogs of the subunit B. In cyanobacteria, a homodimer of subunit B is replaced by a 218 heterodimer of subunit B and B′ (Dunn et al., 2001). However, atpF′F is found in 219 nonphotosynthetic Acidobacteria, e.g., Ca. K. versatilis and Ca. S. usitatus, and also in the 220 photoheterotroph Chloracidobacterium thermophilum.

221 A second function is attributed to terminal oxidases in some organisms, i.e., defence against 222 oxidative stress, especially to bd-type oxidases (Giuffrè et al., 2014). The strictly anaerobic SRM 223 Desulfovibrio vulgaris encodes two terminal oxidases, one cytochrome-c oxidase (HCO class A, 224 DVU_1815–1812) and one bd-type oxidase (DVU_3271–3270). It was demonstrated that both are 225 involved in the detoxification of oxygen (Ramel et al., 2013). Terminal oxidases are also needed 226 to remove oxygen produced by superoxide detoxification. Superoxide dismutase converts 227 superoxide to oxygen and hydrogen peroxide. Hydrogen peroxide is then removed by catalases, 228 peroxidases, or glutathione peroxidases (Figure 3). Manganese-dependent superoxide dismutase 229 (sodA) is found in all MAGs and Cu-Zn-dependent superoxide dismutase (sodC) in SbA2 and 230 SbA7. SbA1, SbA2, SbA3, and SbA5 encode for bifunctional haem-dependent catalase- 231 peroxidases (katG), but none of the seven MAGs for mono-functional, haem-dependent 232 catalases (katE/katA) or manganese-dependent catalases (katN). SbA2 and SbA4 encode for 233 glutathione peroxidases (Supplementary Table S2i).

234 Dissimilatory nitrogen metabolism and nitrogen fixation 235 Although nitrate availability is limited in wetlands (Pester et al., 2012a), we investigated the 236 possibility for nitrate respiration in the MAGs. SRM contribute to nitrogen cycling, as some can 237 respire nitrate/nitrite as an alternative electron acceptor to sulfate or fix atmospheric nitrogen 238 using nitrogenase (Rabus et al., 2013; Marietou, 2016). Oxidation of sulfur compounds coupled 239 to nitrate or nitrite reduction is common among sulfide-/thiosulfate-oxidizing microorganisms 240 (Ghosh and Dam, 2009), but was also observed in organisms encoding reductive-type DsrAB 241 genes. Desulfovibrio desulfuricans, Desulfobulbus propionicus, and Desulfurivibrio alkaliphilus 242 were shown to oxidize sulfide with nitrate/nitrite as the electron acceptor (Dannenberg et al., 243 1992; Thorup et al., 2017). It is also proposed that the sulfide-oxidizing cable bacteria 244 (Desulfobulbaceae) can use nitrate/nitrate as an alternative to oxygen (Marzocchi et al., 2014).

245 Only few Acidobacteria were shown to perform nitrate reduction or encode the required marker 246 genes (e.g., Ward et al., 2009; Männistö et al., 2012). SbA1–7 lack narGHI, napAB, nrfA, nirK, 247 nirS, norBC, and nosZ (Kraft et al., 2011) and thus the genomic potential for dissimilatory nitrate 248 or nitrite reduction. Only SbA5 harbours two gene copies of the nitric oxide reductase NorZ (also 249 known as qNOR), an enzyme that is likely not involved in denitrification but used for nitric oxide

253 Dissimilatory metal reduction 254 The genes required for dissimilatory metal reduction (mtr/omc operon) as described for 255 Shewanella and Geobacter (Shi et al., 2006; Weber et al., 2006; Coursolle and Gralnick, 2010) 256 are absent in all MAGs. Direct interspecies electron transfer (DIET) is an important but 257 understudied process in wetlands (Holmes et al., 2017). However, we could not identify any 258 homologs to the essential pilin-associated c-type cytochrome OmcS (Shrestha et al., 2013; 259 Holmes et al., 2017). We found an ortholog to a novel metal reduction complex in 260 Desulfotomaculum reducens (Dred_1685–1686) (Otwell et al., 2015) in SbA2 (Sbm_v1_c100009– 261 10). This complex was shown to reduce Fe(III), Cr(VI), and U(VI) with NADH as the electron 262 donor, but its physiological role is unresolved (Otwell et al., 2015).

263 Import and phosphorylation of glucose 264 All genomes except SbA7 harbour at least one cytoplasmic glucokinase (glk/glcK) 265 (Supplementary Table S2m). Cytoplasmic glucokinases are required to utilize glucose released 266 by cytoplasmic polysaccharides degradation, but are not required for growth on glucose, as 267 extracellular glucose can be imported and phosphorylated by the phosphotransferase system 268 (PTS). The PTS is missing in all MAGs — Enzyme I and histidine protein are both not present. 269 Only one fragment of a mannitol-specific enzyme IIBC component is found in SbA2 270 (Sbm_v1_c130007). Alternately, Lindner et al. (2011) demonstrated that inositol permeases 271 IolT1/IolT2 are low-affinity glucose permeases, and together with glucokinases can replace the 272 PTS in Corynebacterium glutamicum. iolT1 and iolT2 match TIGRFAM’s sugar porter motif 273 TIGR00879, which we also find in genes in all genomes but SbA2 (data not shown). These could 274 putatively transport glucose, but also other sugars or inositol.

275 N-acetylgalactosamine degradation 276 N-acetylgalactosamine (GalNAc) degradation consists of five steps before entering glycolysis: 277 (1) N-acetylgalactosamine kinase, (2) N-acetylgalactosamine-6-phosphate deacetylase, (3) 278 galactosamine-6-phosphate deaminase, (4) tagatose-6-phosphate kinase, and (5) tagatose- 279 bisphosphate aldolase (Figure 3, Supplementary Table S2l). Neither GalNAc-specific PTS genes 280 or N-acetylgalactosamine kinase (agaK) were identified. However, other sugar kinases of are 281 present, e.g., glcK-type glucokinases. Not all sugar kinases are specific for only one substrate 282 (e.g., Reith et al., 2011), therefore some of the those could putatively act as N- 283 acetylgalactosamine kinases. N-acetylglucosamine-6-phosphate deacetylase (nagA) was found 284 in six MAGs. E. coli possesses two homologous N-acetylglucosamine-6-phosphate deacetylases

295 Lactate, propionate, and butyrate metabolism 296 Six of the acidobacterial MAGs harbour four different types of L-lactate dehydrogenases, while 297 SbA6 has none (Supplementary Table S2o). NAD-dependent L-lactate dehydrogenase Ldh (SbA7 298 and SbA5) ferments pyruvate to L-lactate anaerobically, while the FMN-dependent L-lactate 299 dehydrogenase LldD (SbA1, SbA7, SbA3, and SbA4), L-lactate/D-lactate/glycolate 300 dehydrogenase GlcDEF (SbA5, SbA3, and SbA4) and, LUD-type L-lactate dehydrogenase LutABC 301 (SbA5, SbA2, SbA3, and SbA4) utilize L-lactate as an energy and carbon source. We found 302 putative D-lactate dehydrogenases (Dld) (SbA1, SbA5, and SbA2), which probably convert D- 303 lactate to pyruvate. These are homologs to Archaeoglobus fulgidus Dld (AF_0394) and 304 mitochondrial D-lactate dehydrogenases (<30% identity).

305 With the exception of SbA2, all MAGs contain key genes for propionate oxidation with complete 306 pathways found in SbA1 and SbA5 (Supplementary Table S2o). Conversion of propionate to 307 propionyl-CoA is performed by a CoA transferase. Propionyl-CoA:succinate CoA transferase ScpC 308 is encoded in SbA1 and acetate CoA-transferase YdiF, a family 1 CoA transferase, which has 309 propionyl-CoA:acetate CoA transferase activity (Rangarajan et al., 2005), is encoded in SbA5 310 and SbA4 (Supplementary Table S2o). Other family 1 CoA transferase genes (IPR004165) are 311 present,except in SbA2. It is unclear if these can utilize propionate as well, however it was 312 proposed before for Desulfotomaculum kuznetsovii (Visser et al., 2013). The main subunit gene 313 of the propionyl-CoA carboxylase (PccB), which produces (S)-methylmalonyl-CoA, is present in 314 all six MAGs. The propionyl-CoA carboxylase biotin carboxylase subunit and biotin carboxyl 315 carrier protein are, however, missing in SbA7 and SbA4. Stereochemical inversion to (R)- 316 methylmalonyl-CoA is performed by methylmalonyl-CoA epimerase (Mce), which is encoded in 317 the same MAGs as ScpC/YdiF. Methylmalonyl-CoA mutase, catalyzing the final step in the 318 pathway, is present in all six MAGs (Supplementary Table S2o).

319 Various putative beta-oxidation genes are present in all MAGs, however the substrate 320 specificities of their encoded enzymes are unclear. For the case of butyrate oxidation, the

9 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 321 physiological mechanisms are resolved in detail in the syntrophic organism Syntrophomonas 322 wolfei (Schmidt et al., 2013). No orthologs to the key enzyme butyryl-CoA dehydrogenase 323 (Swol_1933/Swol_2052) are present in any of the MAGs, therefore it is unlikely they can perform 324 (syntrophic) butyrate oxidation.

325 Differential gene expression between anoxic microcosm incubations 326 To analyze changes in gene expression of SbA1–7 during the anoxic peat soil incubations, we 327 performed pairwise comparisons between different treatments and time points: (1) at every 328 time point and for each added substrate we compared the microcosms amended with sulfate to 329 those without external sulfate (i.e., stimulation or downregulation caused by sulfate), (2) at 330 every time point and separately for sulfate-stimulated incubations and no-sulfate-controls we 331 compared microcosms amended with substrate to the no-substrate-controls (i.e., up- or 332 downregulation caused by formate, acetate, propionate, lactate, or butyrate); and (3) for each 333 treatment we compared the early time point (8 days) to the late time point (36 days) 334 (Supplementary Table S3a). When compared to the gene expression changes between the 335 native soil and the incubations, less genes were upregulated between different incubations 336 treatments. Differentially expressed genes included dissimilatory sulfur genes, hydrogen 337 metabolism genes, electron transfer genes, and a few genes belonging to the tricarboxylic acid 338 cycle (Supplementary Table S3a).

339 Compared to the butyrate-only incubation, expression of sat, aprBA, qmoBC, dsrAB, dsrN, dsrT, 340 and dsrL of SbA2 was induced upon addition of sulfate and butyrate. One subunit of (2R)- 341 sulfolactate sulfo-lyase (suyB) was overexpressed in SbA4 in incubations with sulfate and 342 formate. Compared to the no-substrate-controls, we observed significant overexpression of 343 some sulfur metabolism genes from SbA2, SbA3, and SbA7 in formate-, propionate-, lactate-, 344 and/or butyrate-amended incubations, with and/or without supplemental sulfate 345 (Supplementary Table S3a). However, we observed no significant expression changes in the 346 genes that are possibly involved in oxidation of the amended substrates. Moderate expression 347 of sulfate reduction genes without addition of external sulfate is expected due to cryptic sulfur 348 cycling under anoxic conditions (Pester et al., 2012a). The peat soil microcosms without external 349 sulfate contained low amounts of endogenous sulfate (24±6 µM) that was only depleted after 350 11–25 days of incubation (Hausmann et al., 2016).

351 Group 3 hydrogenase gene were affected by substrate amendment in incubation with and/or 352 without external sulfate added. Group 3b hydrogenase (hyhBCSL) of SbA2 was stimulated by all 353 substrates except acetate. Group 3c hydrogenase (mvhDCA) of SbA4 was significantly 354 downregulated after the addition of propionate, lactate, and butyrate. Group 3d hydrogenase 355 (hoxEFYH) of SbA1 was significantly overexpressed in butyrate-amended microcosms. HoxF of 356 SbA2 was overexpressed when lactate or butyrate was added. Group 3 hydrogenases are

10 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 357 cytoplasmic and possibly bidirectional (Greening et al., 2016), leaving it unresolved if hydrogen 358 is produced from the substrates or if hydrogen is provided from a substrate-utilizing syntrophic 359 partner.

360 Supplementary References 361 Babul J. (1978). Phosphofructokinases from Escherichia coli: purification and characterization of 362 the nonallosteric isozyme. J Biol Chem 253: 4350–4355.

363 Buggy J, Bauer CE. (1995). Cloning and characterization of senC, a gene involved in both aerobic 364 respiration and photosynthesis gene expression in Rhodobacter capsulatus. J Bacteriol 177: 365 6958–6965.

366 Bühler D, Rossmann R, Landolt S, Balsiger S, Fischer H-M, Hennecke H. (2010). Disparate 367 pathways for the biogenesis of cytochrome oxidases in Bradyrhizobium japonicum. J Biol Chem 368 285: 15704–15713.

369 Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y et al. (2014). Ribosomal Database Project: 370 data and tools for high throughput rRNA analysis. Nucleic Acids Res 42: D633–D642.

371 Cook GM, Poole RK. (2016). A bacterial oxidase like no other? Science 352: 518–519.

372 Coursolle D, Gralnick JA. (2010). Modularity of the Mtr respiratory pathway of Shewanella 373 oneidensis strain MR-1. Mol Microbiol 77: 995–1008.

374 Dahl C, Friedrich CG. (2008). Microbial Sulfur Metabolism. Springer, Berlin Heidelberg.

375 Daims H, Lücker S, Wagner M. (2016). A new perspective on microbes formerly known as nitrite- 376 oxidizing bacteria. Trends Microbiol 24: 699–712.

377 Dannenberg S, Kroder M, Dilling W, Cypionka H. (1992). Oxidation of H₂, organic compounds and 378 inorganic sulfur compounds coupled to reduction of O₂ or nitrate by sulfate-reducing bacteria. 379 Arch Microbiol 158: 93–99.

380 Dibrova DV, Galperin MY, Mulkidjanian AY. (2010). Characterization of the N-ATPase, a distinct, 381 laterally transferred Na⁺-translocating form of the bacterial F-type membrane ATPase. 382 Bioinformatics 26: 1473–1476.

383 Dunn SD, Kellner E, Lill H. (2001). Specific heterodimer formation by the cytoplasmic domains of 384 the b and b′ subunits of cyanobacterial ATP synthase. Biochemistry 40: 187–192.

385 Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ et al. (2002). The complete

388 Friedrich T, Dekovic DK, Burschel S. (2016). Assembly of the Escherichia coli NADH:ubiquinone 389 oxidoreductase (respiratory complex I). Biochim Biophys Acta 1857: 214–223.

390 Gaby JC, Buckley DH. (2014). A comprehensive aligned nifH gene database: a multipurpose tool 391 for studies of nitrogen-fixing bacteria. Database (Oxford) 2014: bau001.

392 Garcia Costas AM, Liu Z, Tomsho LP, Schuster SC, Ward DM, Bryant DA. (2012). Complete 393 genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based 394 photoheterotroph belonging to the phylum Acidobacteria. Environ Microbiol 14: 177–190.

395 Ghosh W, Dam B. (2009). Biochemistry and molecular biology of lithotrophic sulfur oxidation by 396 taxonomically and ecologically diverse bacteria and archaea. FEMS Microbiol Rev 33: 999–1043.

397 Giuffrè A, Borisov VB, Arese M, Sarti P, Forte E. (2014). Cytochrome bd oxidase and bacterial 398 tolerance to oxidative and nitrosative stress. Biochim Biophys Acta 1837: 1178–1187.

399 Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB et al. (2016). Genomic and 400 metagenomic surveys of hydrogenase distribution indicate H₂ is a widely utilised energy source 401 for microbial growth and survival. ISME J 10: 761–777.

402 Guest JR. (1981). Partial replacement of succinate dehydrogenase function by phage- and 403 plasmid-specified fumarate reductase in Escherichia coli. J Gen Microbiol 122: 171–179.

404 Hausmann B, Knorr K-H, Schreck K, Tringe SG, Glavina del Rio T, Loy A et al. (2016). Consortia of 405 low-abundance bacteria drive sulfate reduction-dependent degradation of fermentation 406 products in peat soil microcosms. The ISME Journal 10: 2365–2375.

407 Holmes DE, Shrestha PM, Walker DJF, Dang Y, Nevin KP, Woodard TL et al. (2017). 408 Metatranscriptomic evidence for direct interspecies electron transfer between Geobacter and 409 Methanothrix species in methanogenic rice paddy soils. Appl Environ Microbiol 83: e00223–17.

410 Hu Z, Patel IR, Mukherjee A. (2013). Genetic analysis of the roles of agaA, agaI, and agaS genes 411 in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli 412 strains O157:H7 and C. BMC Microbiol 13: 94.

413 Huang CJ, Barrett EL. (1991). Sequence analysis and expression of the Salmonella typhimurium 414 asr operon encoding production of hydrogen sulfide from sulfite. J Bacteriol 173: 1544–1553.

415 Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D, Walter MC et al. (2016). eggNOG 4.5:

418 Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. (2010). Prodigal: prokaryotic 419 gene recognition and translation initiation site identification. BMC Bioinformatics 11: 119.

420 Iguchi H, Yurimoto H, Sakai Y. (2010). Soluble and particulate methane monooxygenase gene 421 clusters of the type I methanotroph Methylovulum miyakonense HT12. FEMS Microbiol Lett 312: 422 71–76.

423 Johnson EF, Mukhopadhyay B. (2005). A new type of sulfite reductase, a novel coenzyme F₄₂₀- 424 dependent enzyme, from the methanarchaeon Methanocaldococcus jannaschii. J Biol Chem 425 280: 38776–38786.

426 Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C et al. (2014). InterProScan 5: genome- 427 scale protein function classification. Bioinformatics 30: 1236–1240.

428 Katoh K, Standley DM. (2013). MAFFT multiple sequence alignment software version 7: 429 improvements in performance and usability. Mol Biol Evol 30: 772–780.

430 Klenk HP, Clayton RA, Tomb JF, White O, Nelson KE, Ketchum KA et al. (1997). The complete 431 genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus 432 fulgidus. Nature 390: 364–370.

433 Kraft B, Strous M, Tegetmeyer HE. (2011). Microbial nitrate respiration–genes, enzymes and 434 environmental distribution. J Biotechnol 155: 104–117.

435 Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. (2001). Predicting transmembrane protein 436 topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567– 437 580.

438 Kuever J, Visser M, Loeffler C, Boll M, Worm P, Sousa DZ et al. (2014). Genome analysis of

439 Desulfotomaculum gibsoniae strain GrollT a highly versatile Gram-positive sulfate-reducing 440 bacterium. Stand Genomic Sci 9: 821–839.

441 Langmead B, Salzberg SL. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9: 442 357–359.

443 Lartillot N, Lepage T, Blanquart S. (2009). PhyloBayes 3: a Bayesian software package for 444 phylogenetic reconstruction and molecular dating. Bioinformatics 25: 2286–2288.

445 Laska S, Lottspeich F, Kletzin A. (2003). Membrane-bound hydrogenase and sulfur reductase of

448 Letunic I, Bork P. (2016). Interactive tree of life (iTOL) v3: an online tool for the display and 449 annotation of phylogenetic and other trees. Nucleic Acids Res 44: W242–W245.

450 Liao Y, Smyth GK, Shi W. (2014). featureCounts: an efficient general purpose program for 451 assigning sequence reads to genomic features. Bioinformatics 30: 923–930.

452 Lindner SN, Seibold GM, Henrich A, Krämer R, Wendisch VF. (2011). Phosphotransferase system- 453 independent glucose utilization in Corynebacterium glutamicum by inositol permeases and 454 glucokinases. Appl Environ Microbiol 77: 3571–3581.

455 Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. (2014). The carbohydrate- 456 active enzymes database (CAZy) in 2013. Nucleic Acids Res 42: D490–D495.

457 Love MI, Huber W, Anders S. (2014). Moderated estimation of fold change and dispersion for 458 RNA-seq data with DESeq2. Genome Biol 15: 550.

459 Maklashina E, Berthold DA, Cecchini G. (1998). Anaerobic expression of Escherichia coli 460 succinate dehydrogenase: functional replacement of fumarate reductase in the respiratory 461 chain during anaerobic growth. J Bacteriol 180: 5989–5996.

462 Mardanov AV, Panova IA, Beletsky AV, Avakyan MR, Kadnikov VV, Antsiferov DV et al. (2016). 463 Genomic insights into a new acidophilic, copper-resistant Desulfosporosinus isolate from the 464 oxidized tailings area of an abandoned gold mine Lueders T (ed). FEMS Microbiol Ecol 92: 465 fiw111.

466 Marietou A. (2016). Nitrate reduction in sulfate-reducing bacteria. FEMS Microbiol Lett 363: 467 fnw155.

468 Marzocchi U, Trojan D, Larsen S, Meyer RL, Revsbech NP, Schramm A et al. (2014). Electric 469 coupling between distant nitrate reduction and sulfide oxidation in marine sediment. ISME J 8: 470 1682–1690.

471 Männistö MK, Rawat S, Starovoytov V, Häggblom MM. (2012). Granulicella arctica sp. nov., 472 Granulicella mallensis sp. nov., Granulicella tundricola sp. nov. and Granulicella sapmiensis sp. 473 nov., novel acidobacteria from tundra soil. Int J Syst Evol Microbiol 62: 2097–2106.

474 Mitchell A, Chang H-Y, Daugherty L, Fraser M, Hunter S, Lopez R et al. (2015). The InterPro 475 protein families database: the classification resource after 15 years. Nucleic Acids Res 43: 476 D213–D221.

479 Müller AL, Kjeldsen KU, Rattei T, Pester M, Loy A. (2015). Phylogenetic and environmental 480 diversity of DsrAB-type dissimilatory (bi)sulfite reductases. ISME J 9: 1152–1165.

481 Notredame C, Higgins DG, Heringa J. (2000). T-Coffee: a novel method for fast and accurate 482 multiple sequence alignment. J Mol Biol 302: 205–217.

483 Otwell AE, Sherwood RW, Zhang S, Nelson OD, Li Z, Lin H et al. (2015). Identification of proteins 484 capable of metal reduction from the proteome of the Gram-positive bacterium 485 Desulfotomaculum reducens MI-1 using an NADH-based activity assay. Environ Microbiol 17: 486 1977–1990.

487 Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. (2015). CheckM: assessing the 488 quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome 489 Res 25: 1043–1055.

490 Pereira IAC, Ramos AR, Grein F, Marques MC, Marques da Silva S, Venceslau SS. (2011). A 491 comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea. 492 Front Microbiol 2: 69.

493 Pereira MM, Santana M, Teixeira M. (2001). A novel scenario for the evolution of haem-copper 494 oxygen reductases. Biochim Biophys Acta 1505: 185–208.

495 Pester M, Bittner N, Deevong P, Wagner M, Loy A. (2010). A ’rare biosphere’ microorganism 496 contributes to sulfate reduction in a peatland. ISME J 4: 1591–1602.

497 Pester M, Knorr K-H, Friedrich MW, Wagner M, Loy A. (2012a). Sulfate-reducing microorganisms 498 in wetlands – fameless actors in carbon cycling and climate change. Front Microbiol 3: 72.

499 Pester M, Maixner F, Berry D, Rattei T, Koch H, Lücker S et al. (2014). NxrB encoding the beta 500 subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing 501 Nitrospira. Environ Microbiol 16: 3055–3071.

502 Pester M, Rattei T, Flechl S, Gröngröft A, Richter A, Overmann J et al. (2012b). amoA-based 503 consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from 504 soils of four different geographic regions. Environ Microbiol 14: 525–539.

505 Plugge CM, Henstra AM, Worm P, Swarts DC, Paulitsch-Fuchs AH, Scholten JCM et al. (2012).

506 Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOBT). Stand Genomic 507 Sci 7: 91–106.

510 Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P et al. (2013). The SILVA ribosomal 511 RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 512 41: D590–D596.

513 R Core Team. (2017). R: a language and environment for statistical computing. R Foundation for 514 Statistical Computing: Vienna, Austria. http://www.r-project.org/.

515 Rabus R, Hansen TA, Widdel F. (2013). Dissimilatory sulfate- and sulfur-reducing prokaryotes. In: 516 Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds). The Prokaryotes – 517 prokaryotic physiology and biochemistry. Springer Berlin Heidelberg, pp 309–404.

518 Rabus R, Ruepp A, Frickey T, Rattei T, Fartmann B, Stark M et al. (2004). The genome of 519 Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic 520 sediments. Environ Microbiol 6: 887–902.

521 Ramel F, Amrani A, Pieulle L, Lamrabet O, Voordouw G, Seddiki N et al. (2013). Membrane- 522 bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris 523 Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation. 524 Microbiology 159: 2663–2673.

525 Rangarajan ES, Li Y, Ajamian E, Iannuzzi P, Kernaghan SD, Fraser ME et al. (2005). 526 Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA 527 transferases. J Biol Chem 280: 42919–42928.

528 Refojo PN, Teixeira M, Pereira MM. (2012). The Alternative complex III: properties and possible 529 mechanisms for electron transfer and energy conservation. Biochim Biophys Acta 1817: 1852– 530 1859.

531 Reith J, Berking A, Mayer C. (2011). Characterization of an N-acetylmuramic acid/N- 532 acetylglucosamine kinase of Clostridium acetobutylicum. J Bacteriol 193: 5386–5392.

533 Schmidt A, Müller N, Schink B, Schleheck D. (2013). A proteomic view at the biochemistry of 534 syntrophic butyrate oxidation in Syntrophomonas wolfei. PLoS One 8: e56905.

535 Shi L, Chen B, Wang Z, Elias DA, Mayer MU, Gorby YA et al. (2006). Isolation of a high-affinity 536 functional protein complex between OmcA and MtrC: two outer membrane decaheme c-type 537 cytochromes of Shewanella oneidensis MR-1. J Bacteriol 188: 4705–4714.

538 Shrestha PM, Rotaru A-E, Summers ZM, Shrestha M, Liu F, Lovley DR. (2013). Transcriptomic and

16 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 539 genetic analysis of direct interspecies electron transfer. Appl Environ Microbiol 79: 2397–2404.

540 Simon J, Kroneck PMH. (2013). Microbial sulfite respiration.

541 Strittmatter AW, Liesegang H, Rabus R, Decker I, Amann J, Andres S et al. (2009). Genome 542 sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic 543 carbon completely to carbon dioxide. Environ Microbiol 11: 1038–1055.

544 Sumi M, Yohda M, Koga Y, Yoshida M. (1997). F₀F₁-ATPase genes from an archaebacterium, 545 Methanosarcina barkeri. Biochem Biophys Res Commun 241: 427–433.

546 The UniProt Consortium. (2015). UniProt: a hub for protein information. Nucleic Acids Res 43: 547 D204–D212.

548 Thorup C, Schramm A, Findlay AJ, Finster KW, Schreiber L. (2017). Disguised as a sulfate 549 reducer: growth of the deltaproteobacterium Desulfurivibrio alkaliphilus by sulfide oxidation with 550 nitrate. MBio 8: e00671–17.

551 Vallenet D, Calteau A, Cruveiller S, Gachet M, Lajus A, Josso A et al. (2017). MicroScope in 2017: 552 an expanding and evolving integrated resource for community expertise of microbial genomes. 553 Nucleic Acids Res 45: D517–D528.

554 Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, Mavrommatis K, Kyrpides NC et al. 555 (2015). Microbial species delineation using whole genome sequences. Nucleic Acids Res 43: 556 6761–6771.

557 Visser M, Worm P, Muyzer G, Pereira I a C, Schaap PJ, Plugge CM et al. (2013). Genome analysis

558 of Desulfotomaculum kuznetsovii strain 17T reveals a physiological similarity with

559 Pelotomaculum thermopropionicum strain SIT. Stand Genomic Sci 8: 69–87.

560 Ward NL, Challacombe JF, Janssen PH, Henrissat B, Coutinho PM, Wu M et al. (2009). Three 561 genomes from the phylum Acidobacteria provide insight into the lifestyles of these 562 microorganisms in soils. Appl Environ Microbiol 75: 2046–2056.

563 Wasmund K, Mußmann M, Loy A. (2017). The life sulfuric: microbial ecology of sulfur cycling in 564 marine sediments. Environ Microbiol Rep. doi:https://doi.org/10.1111/1758- 565 2229.12538[10.1111/1758-2229.12538].

566 Weber KA, Achenbach LA, Coates JD. (2006). Microorganisms pumping iron: anaerobic microbial 567 iron oxidation and reduction. Nat Rev Microbiol 4: 752–764.

568 Weissgerber T, Zigann R, Bruce D, Chang Y-j, Detter JC, Han C et al. (2011). Complete genome

17 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 569 sequence of Allochromatium vinosum DSM 180T. Stand Genomic Sci 5: 311–330.

570 Wu AJ, Penner-Hahn JE, Pecoraro VL. (2004). Structural, spectroscopic, and reactivity models for 571 the manganese catalases. Chem Rev 104: 903–938.

572 Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y. (2012). dbCAN: a web resource for automated 573 carbohydrate-active enzyme annotation. Nucleic Acids Res 40: W445–W451.

574 Supplementary Tables

575 Supplementary Table S1 576 Taxonomy, genome characteristics, and abundance measures of the DsrAB-encoding MAGs. 577 Estimation of completeness and contamination was performed with checkM. Genome 578 abundance estimates are the fraction of metagenomic reads mapped to each MAG in relation to 579 all quality filtered reads. Values given for native soil are averages of both native soil 580 metagenomes. mRNA abundance estimates (only acidobacterial MAGs) are the fraction of 581 fragments (paired-end reads) mapped to all of each MAG’s CDS in relation to all quality filtered 582 fragments. Standard deviation of three replicates is given. Fraction of expressed CDS in native 583 soil (%) is given only for acidobacterial MAGs.

584 Supplementary Table S2 585 Curated annotation tables of SbA1–7: (a) dissimilatory sulfur metabolism; (b–h) respiratory 586 complexes I–V: NADH dehydrogenases (NDH, b), succinate dehydrogenase (SDH, c), quinol— 587 cytochrome-c reductases (CIII/ACIII, d/e), low- (LATO, f) and high-affinity (HATO, g) terminal 588 oxidases, and ATP synthases (h); (i) stress (superoxide detoxification), (j) formate 589 dehydrogenases (FDH), hydrogenases (Hase); (k) cytoplasmic electron transport systems; (l) N- 590 acetylgalactosamine degradation; (m) glycolysis (and gluconeogenesis), pentose phosphate 591 pathway, and Entner-Doudoroff pathway; (n) citric acid cycle (TCA); (o) pyruvate, acetate, 592 propionate, and related metabolisms; (p) dissimilatory metal metabolism. Columns provide 593 functional categories (only a, m, o), pathway step number and/or proposed direction (only m, o), 594 product (enzyme, transporter) names with EC and TC numbers where appropriate, subunit 595 names or descriptions where appropriate, gene names, and locus numbers per MAG. All loci are 596 prefixed by Sbm_v1_. Products with multiple copies per MAG are separated into more than one 597 column (only b–f, j). Fragmented genes (assembly or biological artefacts) are marked with 598 downward arrows (↓) after their loci numbers. TM, transmembrane subunit. ¹ or ² indicates the 599 first or last CDS on a scaffold (depending on the reading frame).

600 The following metabolic marker genes are absent in SbA1–7 MAGs: Inorganic sulfur metabolism 601 (Wasmund et al., 2017): tsdA, thiosulfate dehydrogenase; otr, octaheme tetrathionate

18 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 602 reductase; phsABC, thiosulfate reductase; psrABC, polysulfide reductase; sreABC, sulfur 603 reductase (Laska et al., 2003); asrABC, siroheme-independent dissimilatory sulfite reductase 604 (anaerobic sulfite reductase) (Huang and Barrett, 1991); fsr, coenzyme F₄₂₀-dependent sulfite 605 reductase (Johnson and Mukhopadhyay, 2005). ATPases: atpDCQRBEFAG, N-ATPase (alternative 606 “archaeal-type” FₒF₁-ATPase) (Sumi et al., 1997; Dibrova et al., 2010). Dissimilatory nitrate 607 reduction to ammonium (DNRA) (Kraft et al., 2011): napAB, periplasmic nitrate reductase (NAP), 608 catalytic subunit is NapA; nrfA, periplasmic cytochrome c nitrite reductase (catalytic subunit). 609 Denitrification (Kraft et al., 2011): narGHI, membrane bound cytoplasm-facing nitrate reductase 610 (NAR), catalytic subunit is NarG; nirK or nirS, isofunctional but evolutionarily unrelated 611 periplasmic nitric oxide-forming nitrite reductase (NIR); norBC, membrane bound periplasm- 612 facing nitric oxide reductase (NOR), catalytic subunit is NorB; nosZ, periplasmic nitrous oxide 613 reductase (NOS). Nitrogen fixation: nifH, nitrogenase. Nitrification: amoCAB, ammonia 614 monooxygenase (AMO); nxrAB, nitrite oxidoreductase (NXR). Methanotrophy (Iguchi et al., 615 2010): pmoCAB, particulate methane monooxygenase (pMMO); mmoXYBZDC, soluble methane 616 monooxygenase (sMMO). Photosynthesis: pscAB-fmoA, Chloracidobacterium thermophilum 617 photosystem (Garcia Costas et al., 2012); pscABCD, Chlorobium tepidum photosystem (Eisen et 618 al., 2002); pufABCMLH, Allochromatium vinosum photosystem (Weissgerber et al., 2011). ROS 619 defence: katN, mono-functional, manganese catalase (EC 1.11.1.6) (Wu et al., 2004); katE/katA, 620 mono-functional, haem-containing catalase (EC 1.11.1.6). Hydrogenases: [FeFe] hydrogenases.

621 Supplementary Table S3 622 (a) Supplementary Table S2 deposited in machine-readable format including additional 623 information. The length of CDS terminated by scaffold borders (¹ or ² in strand column) is 624 underestimated, as the true length is not known. bactNOG and NOG IDs were assigned by best- 625 match principle. Ranks are based on averaged FPKM. Missing ranks indicate that expression was 626 never detected in any replicate. Significant differential expression is shown separated by factor. 627 Three-letter-codes are initials of amended substrates (F, A, P, L, B) or N for no-substrate-control, 628 sulfate stimulation (S) or control without external sulfate (C), and early (E, 8 days) and late (L, 629 36 days) time points, followed by the log₂ fold change. Over- or underexpression is indicated by 630 arrows of small/larger than signs. (b) Glycoside hydrolase genes identified with dbCAN.

631 Supplementary Table S4 632 Glycoside hydrolases genes summarized by EC numbers provided by the carbohydrate-active 633 enzymes database.

635 Supplementary Figure S1 636 Reductive bacterial-type DsrAB. Maximum likelihood tree was calculated by FastTree (LG model, 637 1000 resamplings) using a reference amino acid alignment with reductive bacterial-type DsrAB 638 indel positions removed (Müller et al., 2015). Branch supports equal to or greater than 0.9 are 639 indicated by black circles. DsrAB sequences from MAGs and scaffolds are marked in bold. 640 Binned acidobacterial DsrAB sequences are coloured analogous to Figure 3. Dashed branches 641 represent incomplete dsrAB gene sequences (e.g., caused by a contig ending) that were 642 sufficiently long to be included in the phylogenetic analysis (only dsrAB genes on scaffold 43ik 643 were too short and omitted). The extent of subdivision 3 group is unclear and indicated by a 644 dashed line. Outgroup sequences are shown in Supplementary Figure S2.

645 Supplementary Figure S2 646 Oxidative bacterial-type DsrAB. Maximum likelihood phylogenetic tree was calculated by 647 FastTree (LG model, 1000 resamplings) using a reference amino acid alignment with oxidative 648 bacterial-type DsrAB indel positions removed (Müller et al., 2015). Branch supports equal to or 649 greater than 0.9 are indicated by black circles. DsrAB sequences from MAGs and scaffolds are 650 marked in bold. Dashed branches represent partial DsrAB sequences on scaffolds.

651 Supplementary Figure S3 652 Phylogenomic tree and pairwise average amino acid identities of Acidobacteria genomes and 653 MAGs. NCBI assembly accessions are given in parentheses. Novel sequences from this study are 654 marked in bold. Acidobacterial subdivisions are given below. Dendrograms are a phylogenomic 655 tree calculated with phylobayes from a checkM-produced and -filtered amino acid alignment. All 656 branches are supported >0.9. Genomes assemblies from the Firmicutes, Proteobacteria, and 657 Verrucomicrobia were used as outgroup. MAG SbA2 has an AAI of 37–49% to the other genomes 658 and MAGs but was not included in the figure because it lacks the marker genes used for the 659 phylogenomic tree.

660 Supplementary Figure S4 661 Beeswarm plots of expression change of dissimilatory sulfur metabolism genes in anoxic peat 662 soil microcosms. Fold-changes are calculated by pairwise comparisons between replicate 663 metatranscriptomes of the native soil and of each incubation regime and time point. Significant 664 (p<0.05) changes are highlighted by back circles.

665

666

20 bioRxivFigure preprint S1. doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Archaeoglobus available under aCC-BY 4.0 International license.

Thermodesulfobium narugense (AB077818, CP002690)

supercluster Nitrospirae Nitrospirae supercluster DsrAB lineages (e.g., Thermodesulfovibrio, uncultured DsrAB lineage 13) Peat soil scaffold 9ana Rasner Möser fen soil clone Rm47 (GU127969)

Peat soil scaffold 6n4w 10 lineage Uncultured

Uncultured bacteria (e.g., AY167472, DQ855242) DsrAB Rice field soil clone Bu-d-13 (JN615158) Rice field soil clone Bu-d-16 (JN615159) Uncultured bacteria (e.g., JN615169, EF065049) Peat soil scaffold 3qhb Schlöppnerbrunnen fen peat soil clone dsrSII_2_201 (GU372068) Peat soil scaffold 5eow Environmental supercluster 1 unclassified family Polluted aquifer clone LGWN11 (EF065087) Aarhus bay sediment clone DSRIV-3 (FM179972) Uncultured DsrAB lineage 12 Uncultured DsrAB lineage 9 Environmental supercluster 1 unclassified family Uncultured bacteria (e.g., GU371967, GU371976) Uncultured bacteria (e.g., JN615149, GU372063) New York peatland soil clone W7 (DQ855251) 1 Subdivision Peatland soil clone (DQ855259) Schlöppnerbrunnen fen peat soil clone dsrSII_5_151 (GU372072) Peat soil MAG SbA5 Peat soil scaffold 7lvv Schlöppnerbrunnen fen peat soil clone dsrSbII-2 (AY167483) Peat soil MAG SbA1 Uncultured bacteria (e.g., GU371972, GU372082)

Uncultured bacteria (e.g., AY167468, GU371962) Environmental supercluster 1 supercluster Environmental

Schlöppnerbrunnen fen peat soil clone dsrSII_5_4 (GU371974) Acidobacteria (uncultured 8) DsrAB lineage Peat soil MAG SbA7 Rasner Möser fen soil clone Rm29 (GU127966) Schlöppnerbrunnen fen peat soil clone dsrSII_2_71 (GU371978) Peat soil scaffold 2oy0 Peat soil scaffold 3kqz Schlöppnerbrunnen fen peat soil clone dsrSII_5_30 (GU371973) Uncultured bacteria (e.g., DQ855252, AY953402) Peat soil scaffold 8uww Peat soil MAG SbA2 Peat soil scaffold 2m2h Aarhus bay sediment clone DSRIV-4 (FM179973) Schlöppnerbrunnen fen peat soil clone dsrSII_2_88 (GU371966) Peat soil scaffold 6mog Peat soil scaffold 75ov Peatland soil clone (DQ855250) New York peatland soil clone W86 (DQ855261) Schlöppnerbrunnen fen peat soil clone dsrSII_2_57 (GU371965) 3 Subdivision Schlöppnerbrunnen fen peat soil clone dsrSII_2_79 (GU371977) Peat soil MAG SbA4 Peat soil scaffold 8p2n Peat soil scaffold 7aia Peatland soil clone (DQ855255) New York peatland soil clone W23 (DQ855257) Peatland soil clone (DQ855249) New York peatland soil clone W63 (DQ855260) Schlöppnerbrunnen fen peat soil clone dsrSII_5_10 (GU371968) Peat soil MAG SbA6 Schlöppnerbrunnen fen peat soil clone dsrSII_5_47 (GU371975) Schlöppnerbrunnen fen peat soil clone dsrSII_2_35 (GU371961) Peat soil scaffold 8mg8 Schlöppnerbrunnen fen peat soil clone dsrSII_5_16 (GU371970) Schlöppnerbrunnen fen peat soil clone dsrSII_2_54 (GU371964) Peat soil MAG SbA3 Schlöppnerbrunnen fen peat soil clone dsrSII_5_146 (GU372071) Schlöppnerbrunnen fen peat soil clone dsrSII_2_109 (GU372081) Schlöppnerbrunnen fen peat soil cloned srSII_2_52 (GU371963) Schlöppnerbrunnen fen peat soil clone dsrSII_5_168 (GU372073) Peat soil scaffold 9142 Schlöppnerbrunnen fen peat soil clone dsrSII_2_159 (GU372066) Schlöppnerbrunnen fen peat soil clone dsrSII_5_81 (GU372070) Other Firmicutes group sensu lato DsrAB families (e.g., uncultured DsrAB lineage 2, Carboxythermus) Pelotomaculum propionicicum (AB154391) Desulfurispora thermophila (NZ_AQWN01000008) Gold mine borehole clone SAW6-30 (AY101581) Desulﬁtibacter alkalitolerans (AY751285) Firmicutes group sensu lato unclassiﬁed family

Desulfotomaculum

sensu lato sensu group Firmicutes Gordonibacter Firmicutes group sensu lato unclassiﬁed family

Desulﬁtobacterium Desulfosporosinus Desulfosporosinus meridiei (NC_018515) Desulfosporosinus youngiae (NZ_CM001441) Desulfosporosinus orientis (AF271767) Desulfosporosinus sp. OT (AGAF01000178) Bioreactor clone WP3-60-DSR4 (JN899156) Desulfosporosinus sp. DB (GU372083) Saltmarsh clone WSMX4 (GU288610) Peat soil MAG SbF1 Desulfosporosinus acidiphilus (CP003639)

Uncultured DsrAB lineage 5 Uncultured DsrAB lineage 6 Other Firmicutes group sensu lato DsrAB families (e.g., uncultured DsrAB lineage 7, Firmicutes group sensu lato unclassiﬁed family)

Firmicutes group sensu lato unclassiﬁed family Desulfobacca Desulfobacca acetoxidans related sequences (e.g., AY167463, FJ748854) acetoxidans Polluted aquifer clone LGWK18 (EF065071) lineage Schlöppnerbrunnen fen peat soil clone dsrSbI-60 (AY167466) Polluted aquifer clone LGWI10 (EF065045) Polluted aquifer clone LGWG08 (EF065024) Polluted aquifer clone LGWG25 (EF065022)

Schlöppnerbrunnen fen peat soil clone dsrSbII-9 (AY167477) supercluster Peat soil scaffold 2q2e Deltaproteobacteria Desulfoarculus baarsii lineage Uncultured family level lineage 1 Other Deltaproteobacteria supercluster DsrAB families (e.g., Thermodesulfobacteraceae, Deltaproteobacteriaceae)

Uncultured bacteria (e.g., JQ304763, AB263154) Desulfoglaeba alkanexedens (GQ843834)

Desulfacinum infernum (AF418194) Syntrophobacteraceae Thermodesulforhabdus norvegica (AF334597) Bu-15-rice field soil (JN615144) 0.1 Rise field soil clone Bu-3 (JN615154) Desulfovirga adipica related sequences (e.g., JN615172, AF334591) Desulforhabdus amnigena (AF337901 Syntrophobacter fumaroxidans (AF418193) Rice field soil clone Rhi-7 (JN615177) Syntrophobacter wolinii related sequences (e.g., JN615162, AF418192) Saltmarsh clone WSMA38 (GU288607) Peat soil MAG SbD1 Schlöppnerbrunnen fen peat soil clone dsrSbII-40 (AY167465) Peat soil scaffold 7zw3 Peat soil MAG SbD2 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Figure S2. available under aCC-BY 4.0 International license.

Morella second DsrAB Candidatus Rokubacteria(XU13_C0051G0022) Peat soil MAG SbV1 Peat soil scaffold 4gr7 reductive archaeal-type DsrAB

0.5 reductive bacterial-type DsrAB Magnetococcus marinus (CP000471) Uncultured SAR324 deltaproteobacterium GBSar324 (JX406431, JX406432) Chlorobi Sulfuricella denitriﬁcans (NZ_BAFJ01000008)

Mine drainage metagenome (CABR010000) Betaproteobacteria Thiobacillus-related sequences (e.g., NC_007404, EU155030) Peat soil scaffold 92uu Peat soil MAG SbB1 Peat soil MAG SbB2 Peat soil scaffold 2xz2 Peat soil scaffold 7I3r Sideroxydans lithotrophicus (CP001965) Freshwater microbial community metagenome (2199352000) Thioalkalivibrio nitratireducens (NC_019902) Gammaproteobacteria Candidatus Riegeria galatelae (HQ689138) Symbiont of Paracatenula ruetzleri (EU155050) Symbiont of Paracatenula sp. 15 (EU155053) Azospirillum halopraeferens (AUCF01000010) Magnetospirillum gryphiswaldense (CU459003) Magnetospirillum sp. SO-1 (AONQ01000031) Magnetospirillum magnetotacticum (AAAP010037) Magnetospirillum magneticum (NC_007626)

Coastal sediment clone ws101A12 (JQ256780) Alphaproteobacteria Coastal sediment clone AK199_D8( JQ256774, JQ256776) Peat soil scaffold 60i6 Wastewater metagenome (APMI01072473) Rhodomicrobium vannielii (CP002292) Intertidal surface sediment clone d1_D1 (HQ191181) Intertidal surface sediment clone d1_E6 (HQ191188) Intertidal surface sediment clone JS24_62 (HQ191214) Intertidal surface sediment clone d1_C3 (HQ191179) Intertidal surface sediment clone d1_B6 (HQ191176) Coastal sediment clone ws085G8 (JQ256788) Intertidal surface sediment clone d1_D3 (HQ191187) Intertidal surface sediment clone d2A6 (HQ191173) Intertidal surface sediment clone d1_G2 (HQ191194) Intertidal surface sediment clone d1_H2 (HQ191197) Intertidal surface sediment clone d3G9 (HQ191191) Intertidal surface sediment clone d2H6 (HQ191195) bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. S3.

100 100 42 40 43 43 41 40 41 39 39 39 41 41 40 41 41 41 41 41 41 40 40 40 40 39 39 40 40 40 40 Thermoanaerobaculum aquaticum (GCF_000687145.1) 90 42 100 60 41 42 40 40 41 39 38 38 41 41 40 40 40 41 40 40 40 40 40 39 39 40 39 39 40 40 40 Geothrix fermentans (GCF_000428885.1) 40 60 100 40 40 40 39 40 37 37 37 40 40 39 39 40 40 39 39 39 39 39 39 39 39 38 39 39 39 39 Holophaga foetida (GCF_000242615.2) 80

43 41 40 100 47 43 42 43 40 39 40 42 43 42 42 43 43 42 42 42 42 42 41 41 41 41 42 42 42 42 Chloracidobacterium thermophilum (GCF_000226295.1) 70 43 42 40 47 100 44 43 44 42 40 41 44 44 43 43 43 44 43 43 43 43 42 42 42 42 42 42 43 42 42 Pyrinomonas methylaliphatogenes (GCF_000820845.2) 60 41 40 40 43 44 100 51 54 50 49 50 46 46 45 46 46 46 46 46 45 45 45 44 45 45 44 46 45 45 46 Acidobacteria bacterium KBS 96 (GCF_000381625.1) 40 40 39 42 43 51 100 51 47 46 48 44 44 44 43 44 44 44 43 43 43 44 43 43 43 43 43 43 44 44 Bryobacter aggregatus (GCF_000702445.1) 50

41 41 40 43 44 54 51 100 61 61 62 47 46 46 45 46 46 46 46 46 45 44 45 45 45 45 46 45 45 46 Solibacter usitatus (GCF_000014905.1) 40 39 39 37 40 42 50 47 61 100 59 60 44 44 44 42 43 44 43 43 43 42 42 42 42 42 42 43 42 42 43 Peat soil MAG SbA3 39 38 37 39 40 49 46 61 59 100 73 43 43 43 41 41 42 41 41 42 40 40 40 40 40 40 41 40 41 41 Peat soil MAG SbA6 30

39 38 37 40 41 50 48 62 60 73 100 44 44 44 43 42 43 42 43 43 41 42 41 42 42 41 43 42 42 42 Peat soil MAG SbA4 20

41 41 40 42 44 46 44 47 44 43 44 100 56 56 50 51 52 51 50 49 49 49 48 48 49 49 50 49 49 50 Koribacter versatilis (GCF_000014005.1) 10 41 41 40 43 44 46 44 46 44 43 44 56 100 63 49 51 52 50 49 49 49 48 48 48 48 48 49 48 49 49 Peat soil MAG SbA1 40 40 39 42 43 45 44 46 44 43 44 56 63 100 50 50 51 49 48 48 48 48 47 47 47 47 49 48 48 49 Peat soil MAG SbA7 0 41 40 39 42 43 46 43 45 42 41 43 50 49 50 100 57 60 58 54 54 55 54 52 52 54 53 56 56 54 55 Acidobacteria bacterium KBS 83 (GCF_000381585.1) 41 40 40 43 43 46 44 46 43 41 42 51 51 50 57 100 61 60 55 55 55 53 52 52 54 53 54 54 54 54 Acidobacterium capsulatum (GCF_000022565.1) 41 41 40 43 44 46 44 46 44 42 43 52 52 51 60 61 100 64 57 56 57 55 54 54 55 54 56 56 56 57 Acidobacterium ailaaui (GCF_000688455.1) 41 40 39 42 43 46 44 46 43 41 42 51 50 49 58 60 64 100 56 55 56 55 53 54 57 54 57 56 56 56 Silvibacterium bohemicum (GCF_001006305.1) 41 40 39 42 43 46 43 46 43 41 43 50 49 48 54 55 57 56 100 63 65 52 51 51 52 51 54 53 52 53 Acidobacteria bacterium URHE0068 (GCF_000620725.1) 41 40 39 42 43 45 43 46 43 42 43 49 49 48 54 55 56 55 63 100 61 51 50 50 52 51 53 52 52 52 Peat soil MAG SbA5 40 40 39 42 43 45 43 45 42 40 41 49 49 48 55 55 57 56 65 61 100 53 52 52 54 52 54 53 54 53 Terracidiphilus gabretensis (GCF_001449115.1) 40 40 39 42 42 45 44 44 42 40 42 49 48 48 54 53 55 55 52 51 53 100 61 60 59 58 59 58 59 59 Terriglobus saanensis (GCF_000179915.2) 40 39 39 41 42 44 43 45 42 40 41 48 48 47 52 52 54 53 51 50 52 61 100 71 57 57 57 57 57 58 Terriglobus roseus (GCF_000265425.1) 40 39 39 41 42 45 43 45 42 40 42 48 48 47 52 52 54 54 51 50 52 60 71 100 57 56 57 56 57 58 Terriglobus sp. TAA 43 (GCF_000800015.1) 39 40 39 41 42 45 43 45 42 40 42 49 48 47 54 54 55 57 52 52 54 59 57 57 100 60 61 60 60 60 Granulicella mallensis (GCF_000178955.2) 39 39 38 41 42 44 43 45 42 40 41 49 48 47 53 53 54 54 51 51 52 58 57 56 60 100 62 60 60 61 Granulicella tundricola (GCF_000178975.2) 40 39 39 42 42 46 43 46 43 41 43 50 49 49 56 54 56 57 54 53 54 59 57 57 61 62 100 67 65 66 Acidobacteria bacterium KBS 89 (GCF_000381605.1) 40 40 39 42 43 45 43 45 42 40 42 49 48 48 56 54 56 56 53 52 53 58 57 56 60 60 67 100 70 73 Edaphobacter aggregans (GCF_000745965.1) 40 40 39 42 42 45 44 45 42 41 42 49 49 48 54 54 56 56 52 52 54 59 57 57 60 60 65 70 100 74 Acidobacteria bacterium TAA 166 (GCF_000421065.1) 40 40 39 42 42 46 44 46 43 41 42 50 49 49 55 54 57 56 53 52 53 59 58 58 60 61 66 73 74 100 Acidobacteria bacterium KBS 146 (GCF_000688615.1) T. aquaticum G. fermentans H. foetida C. thermophilum P. methylaliphatogenes KBS 96 B. aggregatus S. usitatus SbA3 SbA6 SbA4 K. versatilis SbA1 SbA7 KBS 83 A. capsulatum A. ailaaui S. bohemicum URHE0068 SbA5 T. gabretensis T. saanensis T. roseus T G. mallensis G. tundricola KBS 89 E. aggregans TAA 166 KBS 146 . sp. TAA 43

S D 4SD 823 SD 4SD Subdivision 3 Subdivision 1 bioRxiv preprint doi: https://doi.org/10.1101/197269; this version posted October 2, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Fig. S4.

Sulfate activation (or APS oxidation)

4 sat 2 ppa

0 hppA -2

-4

-6 SbA1 SbA7 SbA5 SbA2 SbA3 SbA4 SbA6

APS reduction (or sulﬁte oxidation)

8 aprB

6 aprA

4 qmoA 2 qmoB 0 qmoC -2 adk -4

SbA1 SbA7 SbA5 SbA2 SbA3 SbA4 SbA6

Sulﬁte reduction (or sulﬁde oxidation)

8 dsrA

6 dsrB

4 dsrC dsrM 2 fold-change: soil slurry incubation vs native soil

₂ dsrK 0 log dsrJ -2 dsrO -4 dsrP SbA1 SbA7 SbA5 SbA2 SbA3 SbA4 SbA6

Unclear/unknown function

8 dsrD dsrN 6 dsrV

4 dsrWa

2 dsrM2 dsrK2 0 dsrT

-2 dsrL SbA1 SbA7 SbA5 SbA2 SbA3 SbA4 SbA6