bioRxiv preprint doi: https://doi.org/10.1101/166447; this version posted July 21, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 Title: Dramatic expansion of microbial groups that shape the global sulfur cycle 2 3 Authors: Karthik Anantharaman1*, Sean P. Jungbluth2, Rose S. Kantor3, Adi Lavy1, Lesley A. 4 Warren4, Michael S. Rappé5, Brian C. Thomas1, and Jillian F. Banfield1,6,7* 5 Affiliations: 6 1Department of Earth and Planetary Sciences, Berkeley, CA, USA 7 2DOE Joint Genome Institute, Walnut Creek, CA, USA 8 3Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA 9 4Department of Civil Engineering, University of Toronto, Ontario, CANADA 10 5Hawaii Institute of Marine Biology, University of Hawaii at Manoa, Kaneohe, HI, USA 11 6Department of Environmental Science, Policy, and Management, Berkeley, CA, USA 12 7Earth and Environmental Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, 13 USA 14 15 *Corresponding author 16 Email: [email protected] 17 Address: 307 McCone Hall, Berkeley, CA 94720 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 1 bioRxiv preprint doi: https://doi.org/10.1101/166447; this version posted July 21, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 34 Abstract 35 36 The biogeochemical cycle of sulfur on Earth is driven by microbial sulfate reduction, yet 37 organisms from relatively few lineages have been implicated in this process. Recent 38 studies using functional marker genes have detected abundant, novel dissimilatory 39 sulfite reductases that confer the capacity for microbial sulfate reduction and could do 40 not be affiliated with known organisms. Thus, the identity of a significant fraction of 41 sulfate reducing microbes has remained elusive. Here we report the discovery of the 42 capacity for sulfate reduction in the genomes of organisms from twelve bacterial and 43 archaeal phyla, thereby doubling the number of microbial phyla associated with this 44 process. Eight of the twelve newly identified groups are candidate phyla that lack 45 isolated representatives, a finding only possible given genomes from metagenomes. Two 46 candidate phyla, Candidatus Rokubacteria and Candidatus Hydrothermarchaeota contain 47 the earliest evolved genes. The capacity for sulfate reduction has been laterally 48 transferred in multiple events within some phyla, and a key gene potentially capable of 49 switching sulfur oxidation to sulfate reduction in associated cells has been acquired by 50 putatively symbiotic bacteria. We conclude that functional predictions based on 51 phylogeny will significantly underestimate the extent of sulfate reduction across Earth’s 52 ecosystems. Understanding the prevalence of this capacity is integral to interpreting the 53 carbon cycle because sulfate reduction is often coupled to turnover of buried organic 54 carbon. Our findings expand the diversity of microbial groups associated with sulfur 55 transformations in the environment and motivate revision of biogeochemical process 56 models based on microbial community composition. 57 58 The cycling of sulfur is one of Earth’s major biogeochemical processes. Sulfate reduction may 59 be an early evolved microbial metabolism, given evidence for biological fractionation of sulfur 60 isotopes around 3.5 billion years ago1, and it remains an important metabolic platform for 61 anaerobic life2. In natural ecosystems, human microbiomes and engineered systems, this process 3 4 62 is important because the product hydrogen sulfide (H2S) is toxic , can corrode steel , and sour oil 63 reservoirs5. Overall, sulfate reduction is a primary driver in the carbon cycle, and is responsible 6 64 for conversion of ~30% of the organic carbon flux to CO2 in sedimentary environments . 65 Importantly, the coupling of sulfate reduction to oxidation of H2, small chain fatty acids or other 66 carbon compounds limits the availability of these substrates to other organisms and alters the 67 energetics via syntrophic interactions7. All of these processes also impact methane production. 68 Given the many reasons why the biological conversion of sulfate/sulfite to sulfide is important, it 69 is vital that we understand which organisms can carry out the reactions and the pathways 70 involved. 71 72 Dissimilatory sulfite reductase (dsr) genes confer bacteria and archaea the ability to grow via 73 reduction of sulfite and can function in reverse in some organisms that oxidize sulfur8,9. The 74 phylogenetic distribution of organisms with dsr genes has been considered to be quite limited10. 75 The recent availability of thousands of genomes from organisms belonging to many newly 2 bioRxiv preprint doi: https://doi.org/10.1101/166447; this version posted July 21, 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-NC-ND 4.0 International license. 76 sampled phyla has provided the opportunity to test for the presence of dsr genes in bacteria and 77 archaea that have not previously been associated with dissimilatory sulfur metabolism11. 78 79 Results and Discussion 80 81 We analyzed genomes reconstructed from metagenomic sequence datasets recovered from five 82 distinct terrestrial and marine subsurface environments. The sampling sites included an aquifer 12 13 83 adjacent to the Colorado River, USA , a deep subsurface CO2 geyser in Utah, USA , a deep 84 borehole in Japan14, an acidic sulfide mine waste rock site in Canada15, and deep subseafloor 85 basaltic crustal fluids of the hydrothermally active Juan de Fuca ridge flank in the Pacific 86 Ocean16. We identified dsr genes in 122 near-complete microbial genomes (Supplementary 87 Table 1). Phylogenetic analyses using a set of 16 concatenated ribosomal proteins (RP) and the 88 small subunit ribosomal (SSU) RNA gene show that these genomes belong to organisms from 16 89 distinct phylum-level lineages (Table 1), 12 of which were not known to have dsr genes10. In 90 addition, we identified anaerobic sulfite reductase (asr) genes required for sulfite reduction in 91 two bacterial groups not previously reported to have this capacity17. All of the identified catalytic 92 proteins (DsrA, DsrB, and AsrC) contained all conserved sulfite reductase residues and 93 secondary structure elements for the formation of α helices and β sheets18 (Supplementary Fig. 94 1, Supplementary Fig. 2, Supplementary Fig. 3). 95 96 Given our interest in identifying organisms with the capacity to produce sulfide, we searched 97 genomes for operons that contained genes encoding DsrD. This gene is considered a marker for 98 sulfate reduction because it is absent in bacteria that use the reverse dissimilatory sulfate 99 reduction (reverse-dsr) pathway for sulfur oxidation19. Although the exact function of the DsrD 100 protein is unclear, the presence of winged-helix domains in its structure and its association with 101 other core proteins of the dsr complex (dsrABC) suggest a regulatory role in bacterial sulfate 102 reduction20. We identified 78 genomes that encode at least dsrABCD (Supplementary Fig. 4). A 103 multiple alignment of DsrD sequences confirmed highly conserved residues, indicating that the 104 proteins are likely active (Supplementary Fig. 5). These putative sulfate/sulfite reducing 105 microorganisms affiliate with eight distinct phyla not previously reported to be capable of these 106 processes. Four are phyla with isolated representatives (Acidobacteria, Armatimonadetes, 107 Ignavibacteria, Planctomycetes) and four are considered candidate phyla due to the absence of 108 isolated representatives (Candidatus Zixibacteria, Candidatus Schekmanbacteria, Candidatus 109 Desantisbacteria, Candidatus Lambdaproteobacteria) (Fig. 1). The asr pathway for sulfite 110 reduction was found in members of two candidate phyla, Candidatus Omnitrophica and 111 Candidatus Riflebacteria (Supplementary Fig. 6). 112 113 Surprisingly, we identified dsrD genes in eight genomes of organisms affiliating with 114 Candidatus Falkowbacteria, putatively symbiotic bacteria within the Parcubacteria superphylum 115 of the candidate phyla radiation (CPR)21. There is no indication of the presence of other dsr 3 bioRxiv preprint doi: https://doi.org/10.1101/166447; this version posted July 21, 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-NC-ND 4.0 International license. 116 genes in these genomes. Given the predicted close physical and metabolic interactions between 117 CPR and their hosts, we suggest that this small protein could augment host metabolism, as 118 sometimes occurs with viruses/phage and their hosts22. CPR are common in aquifers where 119 conditions oscillate between oxic and anoxic12. Potentially, populations with the dsrD gene could 120 maintain host function by enabling switching between sulfur oxidation to sulfate reduction as 121 conditions change. The predicted Falkowbacteria