bioRxiv preprint doi: https://doi.org/10.1101/2020.06.03.132969; this version posted June 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Mercury methylation by metabolically versatile and cosmopolitan marine 2 bacteria 3 4 Heyu Lin1, David B. Ascher2,3, Yoochan Myung2,3, Carl H. Lamborg4, Steven J. 5 Hallam5,6, Caitlin M. Gionfriddo7, Kathryn E. Holt8, 9 and John W. Moreau1,10,* 6 7 1School of Earth Sciences, The University of Melbourne, Parkville, VIC 3010, AUS 8 2Structural Biology and Bioinformatics, Department of Biochemistry and Molecular 9 Biology, Bio21 Molecular Science and Biotechnology Institute, The University of 10 Melbourne, Parkville, VIC 3010, AUS 11 3Computational Biology and Clinical Informatics, Baker Heart and Diabetes Institute, 12 PO Box 6492, Melbourne, VIC 3004, AUS 13 4Department of Ocean Sciences, University of California, Santa Cruz, CA 95064, USA 14 5Department of Microbiology and Immunology, University of British Columbia, 15 Vancouver, BC V6T 1Z1, CA 16 6Genome Science and Technology Program, University of British Columbia, 17 Vancouver, BC V6T 1Z4, CA 18 7Biosciences Division, Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 19 37831, USA 20 8Department of Infectious Diseases, Central Clinical School, Monash University, VIC 21 3800, AUS 22 9Department of Infection Biology, London School of Hygiene & Tropical Medicine, 23 London WC1E 7HT, UK 24 10Currently at School of Geographical & Earth Sciences, University of Glasgow, 25 Glasgow G12 8QQ, UK 26 27 *Corresponding author: [email protected] 28 The authors declare no competing interests. 29 30 Running title: Mercury methylation by Marinimicrobia 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.03.132969; this version posted June 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 Abstract 33 Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent 34 neurotoxin in terrestrial and marine food webs. This process requires the gene pair 35 hgcAB, which encodes for proteins that actuate Hg methylation, and has been well 36 described for anoxic environments. However, recent studies report potential MeHg 37 formation in suboxic seawater, although the microorganisms involved remain poorly 38 understood. In this study, we conducted large-scale multi-omic analyses to search for 39 putative microbial Hg methylators along defined redox gradients in Saanich Inlet (SI), 40 British Columbia, a model natural ecosystem with previously measured Hg and MeHg 41 concentration profiles. Analysis of gene expression profiles along the redoxcline 42 identified several putative Hg methylating microbial groups, including Calditrichaeota, 43 SAR324 and Marinimicrobia, with the last by far the most active based on hgc 44 transcription levels. Marinimicrobia hgc genes were identified from multiple publicly 45 available marine metagenomes, consistent with a potential key role in marine Hg 46 methylation. Computational homology modelling predicted that Marinimicrobia 47 HgcAB proteins contain the highly conserved structures required for functional Hg 48 methylation. Furthermore, a number of terminal oxidases from aerobic respiratory 49 chains were associated with several SI putative novel Hg methylators. Our findings thus 50 reveal potential novel marine Hg-methylating microorganisms with a greater oxygen 51 tolerance and broader habitat range than previously recognised. 52 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.03.132969; this version posted June 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 53 Introduction 54 Mercury (Hg), a highly toxic metal, is widespread in the environment from primarily 55 anthropogenic sources, leading to increased public concern over the past few decades. 56 (e.g. Fitzgerald and Clarkson 1991, Hsu-Kim et al 2018, Selin 2009). Methylmercury 57 (MeHg) is recognized as a potent neurotoxin that bioaccumulates through both marine 58 and terrestrial food webs (Lee and Fisher 2017, Selin 2009). With implementation of 59 the Minamata Convention on Mercury (Hsu-Kim et al 2018), better understanding is 60 expected of potential MeHg sources, in the context of global biogeochemical cycles 61 and factors influencing Hg speciation. For example, oxygen gradients in seawater have 62 been observed to expand in response to climate change (Stramma et al 2008), and the 63 resulting impacts on biogeochemical cycles, e.g., carbon, nitrogen, and sulfur, have 64 been studied (e.g., Wright et al 2012). Effects on Hg cycling, however, are rarely 65 considered. 66 67 The environmental transformation of Hg(II) to MeHg is a microbially mediated process, 68 for which the proteins are encoded by the two-gene cluster hgcA and hgcB (Parks et al 69 2013). Possession of the hgcAB gene pair is a predictor for Hg methylation capability 70 (Gilmour et al 2013), and the discovery of hgc has stimulated a search for potential Hg 71 methylating microbes in diverse environments (Podar et al 2015). To date, all 72 experimentally confirmed Hg methylators are anaerobes (Grégoire et al 2018, Podar et 73 al 2015) from: three Deltaproteobacteria clades (sulfate-reducing bacteria, SRB; Fe- 74 reducing bacteria, FeRB); and syntrophic bacteria Syntrophobacterales), one clade 75 belonging to fermentative Firmicutes, and an archaeal clade, Methanomicrobia. These 76 microorganisms are ubiquitous in soils, sediments, seawater, freshwater, and extreme 77 environments, as well as the digestive tracts of some animals (Podar et al 2015). 78 Recently, other hgc carriers, not only from anaerobic but also microaerobic habitats, 79 were discovered using culture-independent approaches, including Chloroflexi, 80 Chrysiogenetes, Spirochaetes (Podar et al 2015), Nitrospina (Gionfriddo et al 2016), 81 and Verrucomicrobia (Jones et al 2019). 82 83 A global Hg survey (Lamborg et al 2014) found a prevalence for MeHg in suboxic 84 waters, especially in regionally widespread oxygen gradients at upper and intermediate 85 ocean depths. Oxygen concentrations are lower there due to a combination of physical 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.03.132969; this version posted June 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 86 and biological forcing effects (Paulmier and Ruiz-Pino 2009, Wright et al 2012). These 87 gradients can exist as permanent features in the water column, impinge on coastal 88 margins, or manifest more transiently (e.g., induced by phytoplankton blooms). As 89 oxygen levels decrease, metabolic energy gets increasingly diverted to alternative 90 electron acceptors, resulting in coupling of other biogeochemical cycles, e.g., C, N, S, 91 Fe, and Mn (Bertagnolli and Stewart 2018, Moore and Doney 2007, Wright et al 2012). 92 Recent findings of microaerophilic microbial Hg methylation potential in sea ice and 93 seawater (Gionfriddo et al 2016, Villar et al 2020) raise the possibility that this process 94 contributes significantly to ocean MeHg biomagnification. 95 96 In this study, we used existing Hg (total) and MeHg concentration data from Saanich 97 Inlet (SI), a seasonally anoxic fjord on the coast of Vancouver Island (British Columbia, 98 Canada) to guide targeted metagenomic and metatranscriptomic analyses of SI seawater 99 from varying depths. Saanich Inlet, as a model natural ecosystem for studying microbial 100 activity along defined oxygen gradients (Hawley et al 2017b), provides an ideal site to 101 search for novel putative Hg methylators in low-oxygen environments. Computational 102 homology modelling was performed to predict the functionality of HgcAB proteins 103 encoded for by putative novel Hg-methylators. Finally, we scanned global 104 metagenomic datasets for recognizable hgcA genes, to reassess the environmental 105 distribution of microbial mercury methylation potential. 106 107 Results and Discussion 108 Hg and MeHg concentrations along redox gradients in SI 109 Concentrations of total dissolved Hg (HgT) and monomethylmercury (MeHg) from 110 filtered SI station “S3” seawater samples were vertical profiled from eight different 111 depths (10 m to 200 m) below sea surface. The concentration of HgT at sea surface was 112 ~0.70 pM and remained nearly constant in seawater above 120 m depth, increasing to 113 1.35 pM and ~10.56 pM at 135 m and 200 m depths, respectively (Figure 1A). MeHg 114 was below detection limit (<0.1 pM) for seawater above 100 m depth, but increased to 115 0.50 pM (17.2% of total Hg) at 150 m depth. However, MeHg then decreased to 0.1 116 pM at 165 m depth, becoming undetectable at 200 m (Figure 1B and 1C). 117 118 MAG (metagenome-assembled genome) reconstruction and putative Hg methylator 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.03.132969; this version posted June 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 119 identification 120 A total of 2088 MAGs with completeness >70% and contamination <5% were 121 recovered from all SI metagenomic datasets (generated across all sampling