This is the accepted version of the journal article: Sanz-Sáez, Isabel; Pereira Garcia, Carla; Bravo, Andrea G.; [et al.]. «Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions». En- vironmental Science and Technology, (March 2022). DOI 10.1021/acs.est.1c05635 This version is available at https://ddd.uab.cat/record/256692 under the terms of the license Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions Isabel Sanz-Sáez1, Carla Pereira García1, Andrea G. Bravo1, Laura Trujillo1, Martí Pla i Ferriol2, Miguel Capilla3, Pablo Sánchez1, Rosa Carmen Rodríguez Martín-Doimeadios4, Silvia G. Acinas1*, Olga Sánchez2*. 1 Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, ICM-CSIC, 08003 Barcelona, Catalunya, Spain 2 Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Research Group in Environmental Engineering (GI2AM), Department of Chemical Engineering, University of Valencia, Av. De la Universitat S/N, 46100, Burjassot, Spain. 4 Environmental Sciences Institute (ICAM), Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071 Toledo, Spain * Correspondence: Olga Sánchez: olga.sanchez@uab.es, tel. (+34) 93 586 8022, FAX (+34) 93 581 2387; Silvia G. Acinas: sacinas@icm.csic.es, tel. (+34) 93 230 5886, FAX (+34) 93 230 9555 1 ABSTRACT 2 3 Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) 4 demethylation is performed by the mer operon, specifically by merA and merB genes 5 respectively, but little is known about the mercury tolerance capacity of marine microorganisms 6 and its prevalence in the ocean. Combining culture-dependent analyses with metagenomic and 7 metatranscriptomic analyses, here, we show that marine bacteria encoding mer genes are 8 widespread and active in the global ocean. We explored the distribution of these genes in 290 9 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different 10 oceanographic regions and depths, and assessed their tolerance to diverse concentrations of 11 Hg2+ and MeHg. In particular, Alteromonas sp. ISS312 strain presented the highest tolerance 12 capacity and a degradation efficiency for MeHg of 98.2% in 24 h. Fragment recruitment 13 analyses of Alteromonas sp. genomes (ISS312 strain and its associated reconstructed 14 metagenome assembled genome MAG-0289) against microbial bathypelagic metagenomes 15 confirm their prevalence in the deep ocean. Moreover, we retrieved 54 merA and 6 merB genes 16 variants related to Alteromonas sp. ISS312 strain from global metagenomes and 17 metatranscriptomes from Tara Oceans. Our findings highlights the biological reductive MeHg 18 degradation as a relevant pathway of the ocean Hg biogeochemical cycle. 19 20 Keywords: mercury, methylmercury, marine bacteria, mercury resistant bacteria, merA, merB, 21 minimum inhibitory concentration (MIC) 22 23 Synopsis 24 Active mercury resistance genes detected in marine cultured bacteria are widely distributed in 25 the ocean, including the bathypelagic zone. 1 26 Introduction 27 28 Mercury (Hg) is one of the most toxic, widespread and worrisome contaminants1,2, and is 29 emitted to the atmosphere by natural sources, such as volcanoes and rock weathering, but 30 particularly by anthropogenic activities. The rising Hg levels since the industrial era, estimated 31 as an increase of 450 % in the atmosphere3, makes the study of its biogeochemical cycle a major 32 concern to the scientific community, and as a consequence, the Minamata convention, a global 33 treaty to protect human, wildlife health and the environment from the adverse effects of mercury 34 by e.g. reducing its atmospheric emissions, was hold in 2013 and entered into force in August 35 20174. 36 37 Emitted elemental (Hg0) and inorganic divalent Hg (Hg2+) can be deposited to land and oceans 38 by wet and dry depositions5,6. Inorganic divalent Hg in the ocean can be then volatilized back 39 again to the atmosphere as Hg0 7, or can be methylated8–12 to methylmercury (MeHg), which 40 bioaccumulates and biomagnifies in aquatic food webs3,13,14. As a consequence, humans are 41 exposed to this neurotoxicant mainly through fish and seafood consumption13,15,16. 42 Methylmercury levels in the oceans vary with depth, and usually, measures are being reported 43 low in open ocean surface waters, maximal in intermediate layers, especially in regions of low- 44 oxygen and near or below the thermoclines (up to 1000 m depth), and low and relatively 45 constant in deeper waters (>1000 m depth)13,17,18. While Hg2+ methylation has been reported to 46 occur in oxic and sub-oxic layers of the water column8,17,19–21 mainly associated with the 47 microbial remineralization of sinking particulate organic matter17,18,22, much less is known about 48 MeHg demethylation and Hg2+ reduction processes in the ocean water column. Although MeHg 49 demethylation and Hg2+ reduction processes can be photochemically mediated23–25, light 50 26 penetration in the ocean water column is limited to 200 m , thereby biological MeHg 51 degradation and Hg reduction processes likely govern in the ocean water column. Moreover, 52 taking into account that different concentrations of MeHg can be found through the ocean water 53 column 20,27–29 , it would be plausible to find microorganisms with Hg detoxification capacity, 54 especially in deep aphotic zones. However, very few studies have unveiled and demonstrated 55 the role of microorganisms in MeHg degradation in the vast deep ocean. 56 57 Biological MeHg demethylation and Hg2+ reduction detoxification processes can be mediated 58 by different biotic processes including reductive reactions mediated by the mer operon30,31, but 59 also oxidative reactions32,33, as well as the recently described MeHg degradation performed by 60 methanotrophic bacteria34. In this study, we will focus only in the study of bacteria carrying the 61 mer operon (i.e. biological reductive MeHg degradation pathways). While the operon can be 62 composed by different sets of genes30,31, the key genes are merA and merB. The first one 1 63 codifies a mercuric reductase and is responsible for the transformation of Hg2+ to the less 64 harmful and volatile Hg0 30. The merB gene encodes an organomercurial lyase enzyme that 65 confers resistance to the organic MeHg form. It is the responsible for its demethylation 66 releasing Hg2+ that will be then reduced to Hg0 by merA gene30. These machineries have been 67 found in numerous microorganisms including aerobic and anaerobic species, although 68 demethylation appears to be predominantly accomplished by aerobic organisms31,35. To date, 69 very few studies have showed the presence of mer genes in oceanic waters, with the exception 70 of some studies in the North Pacific and Arctic Oceans36 and a recent analysis in the global 71 bathypelagic ocean. In order to build this gap in knowledge, the aim of this study was to 72 demonstrate that marine bacteria encoding mer genes could degrade MeHg and to explore its 73 prevalence in the open ocean including deep-ocean waters. For this, we took advantage of the 74 MARINHET37 culture collection, that includes marine bacterial strains from a wide variety of 75 oceanographic regions and depths. We analyzed 290 marine heterotrophic bacteria in order to: 76 (i) detect the presence of merA and merB genes in different oceanographic regions and depths, 77 (ii) assess the tolerance of an important fraction of strains encoding those genes to different 78 concentrations of divalent inorganic Hg (Hg2+) and monomethylmercury (MeHg), (iii) describe 79 the degradation potential for the most tolerant strains, and (iv) explore its prevalence and 80 biogeography patterns across oceans and depths by retrieving merA and merB genes from 81 marine microbial metagenomes and metatranscriptomes from Tara Oceans Expedition38,39. 82 83 Identification of Hg resistant bacteria in contrasting aquatic ecosystems and the assessment of 84 their tolerance to different concentrations of MeHg provide new opportunities to explore the 85 ubiquity and prevalence of marine cultured bacteria with detoxification capacity in the open 86 ocean (i.e. non-contaminated sites). On the other hand, this type of study sets the fundamentals 87 for finding suitable microorganisms to be used for bioremediation strategies. 88 89 Materials and Methods 90 91 Primer design for merA and merB genes of Alteromonas and Marinobacter 92 93 Primers were designed in order to identify mercury resistant bacteria among Alteromonas and 94 Marinobacter strains in a culture collection because: (i) these genera are well known to encode 95 the mer operon40–44 in their genomes, (ii) they are the most common culturable heterotrophic 96 bacteria living in open marine waters all around the world, as they have been isolated from a 97 wide variety of marine environments42,45–50, and in the case of Alteromonas it is one of the most 98 ubiquitous cultured taxa in the ocean37, (iii) we have access to a large number of isolates thanks 99 to the MARINHET culture collection (see Supplementary Information for more details about 2 100 the culture collection). For all these reasons, these two genera were a suitable target to prove 101 that mer genes can be found across different oceanographic regions and depths but also 102 demonstrate the MeHg degrading capability of several strains. 103 104 Specific primer pairs were designed separately for: (i) merA of Alteromonas, (ii) merA of 105 Marinobacter, (iii) merA + merB of Alteromonas, and (iv) merA + merB of Marinobacter based 106 on reference sequences downloaded (in 2016) from the from Integrated Microbial Genomes 107 (IMG) database of the Joint Genome Institute (JGI). See Supplementary Information, 108 Supplementary Figure S1 and Supplementary Table S1 for detailed information. 109 Noteworthy, the primers design in this study are useful for detecting exclusively Alteromonas 110 and Marinobacter and might not be suitable for other taxa. 111 112 DNA extraction and PCR conditions 113 The primers previously designed were used for the screening of merA and merAB Hg resistance 114 genes in all Alteromonas and Marinobacter strains (n=290) available at the MARINHET culture 115 collection in 2016 (n=1313). DNA of all the strains was extracted from 48 h liquid cultures 116 grown in Zobell broth medium (i.e. 5 g peptone, 1 g yeast extract in 750 ml of 30 kDa filtered 117 seawater and 250 ml of Milli-Q water) using the DNeasy Blood & Tissue kit (Qiagen) following 118 the manufacturer’s recommendations. Detailed PCR conditions are described in Supplementary 119 Information. The PCR products were verified and quantified by agarose gel electrophoresis with 120 a standard low DNA mass ladder (Invitrogen). Purification and OneShot Sanger sequencing of 121 merA and merAB gene products was performed by Genoscreen (Lille, France) with both forward 122 and reverse primers. Geneious software v.11.0.551 was used for manual cleaning and quality 123 control of the sequences. 124 Minimum inhibitory concentration experiments 125 126 A total of 73 strains from the 290 isolates previously screened by PCR were subjected to 127 Minimum inhibitory concentration (MIC) assays, including then all strains with positive results 128 for merA and/or merAB presence except one that could not grow again from the cryostock. MIC 129 assays were designed based on previous studies44,52 in order to assess the tolerance of the marine 130 strains to different concentrations of inorganic Hg (as mercury(II) chloride, HgCl2) and organic 131 Hg (as methylmercury chloride, CH3HgCl) and thus, to test the activity of merA and merB genes 132 respectively. A stock solution of HgCl2 was prepared at 500 µM with autoclaved Milli-Q water. 133 Liquid cultures of the strains growing in Zobell broth with an optical density (O.D. at 600nm) of 134 0.1 were placed in 24-well plates and inoculated with 5 µM, 10 µM, 20 µM, 25 µM and 50 µM 3 135 HgCl2. In specific cases growth was observed in all HgCl2 concentrations and further MIC 136 assays were done increasing the final concentrations to 50 µM, 60 µM, 70 µM, 80 µM, 90 µM 137 and 100 µM. The tolerance to CH3HgCl was also tested for the most tolerant strains to HgCl2. In 138 these case, 24-well plates were inoculated with the stock solution to reach final concentrations 139 at 2.5 µM, 5 µM, 10 µM, 15 µM and 20 µM. In all plates a positive control (liquid culture of the 140 strains not amended with CH3HgCl or HgCl2) and a negative control (broth media without 141 bacteria in order to check for possible environmental contamination) were included in the 142 assays. Plates were sealed with parafilm and incubated at room temperature (RT, ~20 ºC) and in 143 the dark for 72 h. Visual examination and O.D. measurements at 600 nm were done in a 24 h 144 period using an automatic plate reader (Infinite ® M200, Tecan) and data was collected using 145 the Magellan™ Data Analysis Software (Tecan Diagnostics®). 146 147 Growth curves 148 149 Growth curves were performed to characterize the growth rates of the most tolerant strain 150 (ISS312) to different concentrations of CH3HgCl. We prepared 200 ml of liquid cultures in 151 Zobell broth supplemented with CH3HgCl at final concentrations of 0 µM (positive control), 1 152 µM, 2.5 µM and 5 µM in triplicates. The initial O.D. at 600 nm of the cultures was 0.05 in order 153 to assure enough concentration of cells for growing. Samples for O.D. measurements and for 154 bacterial cell counts were taken approximately every two hours. O.D. was measured at 600 nm 155 with a spectrophotometer (Varian Cary® 100 UV-Vis) and cells were stained with 4’,6- 156 diamidino-2-phenylindole (DAPI) and counted with an automated microscope Zeiss Axio 157 Imager Z2M53,54 using the automated image analysis software ACME Tool 158 (www.technobiology.ch). Predicted growth curves based on O.D. observations and kinetics 159 values including growth rates (µmax), carrying capacity (k) and lag phase time, were calculated 160 with R package growthcurver v.0.3.055 and GrowthRates v.4.3 software56. For graphical 161 representation, replicates of the different growth curves experiments at several CH3HgCl 162 concentrations were averaged. Hence, mean O.D. and standard deviation was calculated for 163 each time point of the curves. 164 165 From the 1 µM and 5 µM growth curves 2 ml samples were also taken for characterizing 166 CH3HgCl degradation rates at time 0, 6, 12, 24 and 48 h. Besides, in order to check the 167 possibility that CH3HgCl was being abiotically removed, we also measured the CH3HgCl 168 concentrations from samples taken from multiwell plates experiments including different 169 controls (more details in Supplementary Information). Description of Hg species concentrations 170 measurements are also explained in Supplementary Information. 4 171 172 Phylogenetic analyses 173 In order to show that MeHg degradation capacity is strain specific, a phylogeny of the isolates 174 screened by PCR for merA and merAB genes was inferred from their partial 16S rRNA 175 sequences in order to detect a possible clustering between all the positive strains. The closest 176 sequence to each isolate 16S rRNA gene was obtained by BLASTn57 against SILVA v.132 177 database. Alignment of the isolates and reference sequences was performed with MUSCLE 178 from the Geneious software v.11.0.551. The alignment was trimmed to the common 16S rRNA 179 gene fragment covered by both sets of sequences. Phylogeny was constructed using maximum- 180 likelihood inference with RAXML-NG 0.9.058 and the GTR evolutionary model with 181 optimization in the among-site rate heterogeneity model and the proportion of invariant sites 182 (GTR+G+I), and 100 bootstrap replicates. In the same way a phylogenetic tree was constructed 183 with the partial 16S rRNA sequences of the positive isolates only. In this tree the closest match 184 in SILVA v.132 database was also included. Presence of merA and merAB genes, origin of the 185 strains, plus their tolerance to HgCl2 and MeHg were added with Interactive Tree of Life 186 (ITOL)59. Supplementary phylogenetic trees were also constructed using aminoacid sequences 187 of merA and merB genes (see Supplementary Information for details). 188 Fragment recruitment analysis of the genome of strain ISS312 and MAG-0289 in bathypelagic 189 metagenomes 190 191 The abundance of ISS312 across the global bathypelagic ocean was assessed thanks to the 192 sequencing of its complete genome (Supplementary Information). In addition, thanks to a 193 previous study60 we could reconstruct a metagenome assembled genome (MAG-0289) also 194 affiliating to Alteromonas mediterranea species and encoding merA and merB genes. Fragment 195 Recruitment Analysis (FRA) of the ISS312 strain and the MAG-0289 was performed by 196 mapping the metagenomic reads of 58 bathypelagic microbial metagenomes from 32 197 stations60,61 from Malaspina Expedition, including free-living (0.2-0.8 µm) and particle-attached 198 (0.8-20 µm) bacterial communities. Analysis were done with BLASTn v2.7.1+57. Details of 199 FRA are explained in Supplementary Information. 200 201 Detection of merA and merB genes in global metagenomes and metatranscriptomes from Tara 202 Oceans expedition 203 5 204 BLASTp analyses were performed using a conservative e-vale (>1E-100) with the merA and 205 merB sequences of the ISS312 genomes against global prokaryotic metagenomes (metaG) and 206 metatranscriptomes (metaT) available in the Ocean Microbial Reference Gene catalogue V2 207 (OMRGC.v2)39 from Tara Oceans Expedition covering surface, deep chlorophyll maximum 208 (DCM) and mesopelagic layers across oceanographic regions. The generation and annotation of 209 the OMRGC.v2, the profiling of taxonomic of metagenomic and metatranscriptomic 210 composition, and the normalization of metagenomic and metatranscriptomic profiles used for 211 the extraction of the abundances of the merA and merB gene homologous had been thoroughly 212 explained in Salazar et al.39. Analyses could be performed thanks to the Ocean Gene Atlas web 213 resource38 and abundance results from metagenomes and metatranscriptomes were normalized 214 based on the percentage of mapped reads. 215 216 Statistical analyses 217 218 ANOVA tests from the stats package of the R Statistical Software 62 was applied in order to 219 observe if the presence of merA and/or merAB genes in the studied strains was linked to 220 specific oceanographic locations. Further, non-parametric Kruskal-Wallis test, from the stats 221 package of the R Statistical Software62, was applied followed by the post hoc pairwise Wilcox 222 test to see the differences between FRA results in different oceanographic regions and between 223 free-living and particle-attached bacterial communities. To assess significance, all the statistical 224 analyses were set to an alpha value of 0.05. 225 226 Nucleotide accession numbers 227 228 Mercury detoxification genes (merA and merAB) detected in this study through PCR were 229 deposited in GenBank under accession numbers MW273028 - MW273125. Alteromonas 230 mediterranea ISS312 genome was deposited in ENA under study accession number 231 PRJEB46669. 232 233 Results and Discussion 234 235 Presence of merA and merB genes among Alteromonas and Marinobacter strains 236 237 Among the 290 strains of the MARINHET bacterial culture collection37, 244 were 238 taxonomically classified as Alteromonas sp. and 46 strains to Marinobacter sp. 239 (Supplementary Table S2). These strains were isolated from different depths, including the 240 surface, the deep chlorophyll maximum (DCM) and the bathypelagic zone. The strains here 6 241 studied for merAB genes were isolated from different oceanic regions such as the North Western 242 Mediterranean Sea (89), South (101) and North Atlantic (42), Indian (44), Arctic (7) and 243 Southern (7) Oceans and included isolates from photic (160) and aphotic (130) layers (Table 1; 244 Supplementary Figure S2 and Supplementary Tables S3-4). 245 246 The functional screening of the merA and merB genes from the 244 Alteromonas and 46 247 Marinobacter strains revealed that 13.5 % (32 out of 244) and 89.1 % (41 out of 46) of the 248 strains presented only merA, respectively, while only 1.6 % (4 out of 244), and 47.8 % (22 out 249 of 46) presented both merA and merB genes (merAB) (Table 1). No significant differences were 250 found between depths neither between oceans (ANOVA, P-value > 0.05) but in general, we 251 found a higher proportion of positive strains coming from waters of the Southern Ocean (71 % 252 despite the lower number of strains tested) and the South Atlantic Ocean (48 %), followed by 253 those retrieved from the North Atlantic (17 %) and North Western Mediterranean Sea (13 %). 254 255 High variability of mercury tolerance within marine bacteria 256 257 It is unknown whether mercury tolerance is a conservative trait within marine bacterial strains 258 of the same genera. To prove so, MIC experiments were done in all Alteromonas and 259 Marinobacter isolates (73 strains) presenting merA and/or merAB detected by PCR except one 260 isolate that was not able to grow again from the cryostock. First, we tested the tolerance for 261 inorganic mercury (HgCl2) and we observed that 32 Alteromonas and 41 Marinobacter strains 262 displayed different levels of tolerance. MIC values ranged generally from 5 to 50 µM. Around 263 50 % of the Alteromonas and Marinobacter strains tested presented a MIC of 20 µM and one of 264 the isolates stood out as it presented a tolerance to HgCl2 up to 70 µM (Supplementary Table 265 S5). Besides, we tested the tolerance to MeHg amended in form of CH3HgCl for those strains 266 already presenting a tolerance to inorganic mercury above 20 µM and encoding the merB gene. 267 For Alteromonas, three strains taxonomically classified as Alteromonas mediterranea presented 268 a high tolerance to CH3HgCl, growing at concentrations up to 10 µM (Figure 1), but all the 269 tested Marinobacter sp. strains did not show a substantial growth above 2.5 µM of CH3HgCl 270 (Figure 1). 271 272 If we take a look into the phylogenetic trees constructed with the 16S rRNA sequences (Figure 273 1) of the strains presenting merA and/or merAB genes different tolerances were found within the 274 same phylogenetic cluster. For example, within the cluster of Alteromonas mediterranea some 275 strains presented a MIC of 20 µM, while the isolate that presented the highest tolerance (70 µM, 276 ISS312) also belonged to the same Alteromonas species (Figure 1). The same occurred among 277 Marinobacter isolates, where members of the Marinobacter hydrocarbonoclasticus cluster 7 278 presented MIC values ranging from 10 to 50 µM (Supplementary Table S5 and Figure 1). 279 The same pattern can be observed in the merA and merAB aminoacids phylogenies 280 (Supplementary Information and Supplementary Figures S3 and S4). 281 The MIC values heterogeneity within strains belonging to the same phylogenetic cluster, 282 suggested that the level of Hg resistance was strain specific, and we probably retrieved different 283 ecotypes within a same species with different tolerances to Hg. Despite these differences 284 between strains, the tolerances found for inorganic Hg were similar to those found in other 285 studies where Alteromonas44,63–65 and Marinobacter66 genera were also isolated from different 286 marine ecosystems such as hydrothermal vents, estuaries or contaminated sediments. However, 287 to the best of our knowledge, this is the first study addressing the tolerance of Marinobacter 288 spp. and Alteromonas mediterranea isolated from the ocean to MeHg. Hence, we found out that 289 a strain affiliating to Alteromonas mediterranea (ISS312) presented a MIC to inorganic Hg 290 higher than other strains already published, up to 70 µM, and we also determined that it was 291 able to grow in the presence of MeHg, presenting a MIC up to 10 µM, representing a good 292 candidate for future bioremediation studies in highly contaminated areas with both organic and 293 inorganic mercuric compounds. 294 295 Description of the highly tolerant Alteromonas sp. strain ISS312 296 297 Strain ISS312, isolated from South Atlantic bathypelagic waters at 4000 m and classified as 298 Alteromonas mediterranea, displayed the highest tolerance to both HgCl2 (70 µM) and MeHg 299 (10 µM). The growth rates of this strain at different concentrations of MeHg were assessed and 300 included: a control without MeHg (0 µM), 1 µM, 2.5 µM and 5 µM MeHg. Growth curves at 0 301 µM and 1 µM were very similar, as well as between 2.5 µM and 5 µM (Figure 2A). We 302 observed that the major difference between growth curves was the length of the lag phase, 303 where bacteria adapt themselves to the growth conditions. This phenomenon seems to be a 304 common trait for Hg resistant strains in the presence of the toxic compound, as this behavior has 305 been repeatedly observed in different species of Pseudomonas sp., Alcaligenes sp. or Bacillus 306 sp.67–69. However, once the cultures started to grow, their growth rates (µmax) were very similar 307 independently of their initial MeHg concentrations, ranging from 0.10 h-1 in the control to 0.09 308 h-1 at 5 µM. Stationary phase was reached in all concentrations at 80 h, even though at this time 309 cultures at higher concentrations of MeHg seemed to be only entering to the plateau (Figure 310 2A). In addition, their carrying capacity (k), i.e. the maximum population size of a species, was 311 between 1.6 and 1.9 based in O.D. measures, revealing very similar values between tested 312 concentrations, an observation also recurrently reported68,70. Transmission electron microscopy 313 (TEM) observations of the ISS312 cultures growing at 0 µM and 5 µM of MeHg also showed 314 similar morphology and ultrastructure of the cells (Figure 2A). 8 315 316 MeHg concentrations were measured in the isolate exposed to 5 µM at different incubation 317 times. MeHg concentrations were reduced by 36 % (2.6 µM) and 72 % (1.1 µM) (Figure 2B) 318 during the lag phase at 6 h and 12 h, respectively. After 24 h, when almost all MeHg was 319 removed (removal of 98.2 %), the microorganism began the exponential growth phase. After 48 320 h, MeHg could not be detected (Figure 2B Supplementary Table S6). It is important to 321 highlight that we detected a certain level of abiotic MeHg degradation (up to 25%) in the 322 medium exposed without bacteria and in the killed control (Supplementary Table S7). 323 324 Global distribution of ISS312 strain and MAG-0289 in the bathypelagic ocean 325 326 The biogeographic and size fraction distribution of ISS312 genome was assessed in all available 327 bathypelagic metagenomes of the Malaspina Expedition60 since strain ISS312 was originally 328 retrieved from bathypelagic waters of the South Atlantic Ocean. We found that this strain 329 belonging to Alteromonas mediterranea species was distributed across all the temperate 330 bathypelagic waters, including the Atlantic, the Pacific and the Indian Oceans (Figure 3). Its 331 abundances, according to the data from the fragment recruitment analyses (FRA), varied across 332 ocean basins and we found significant differences between the Pacific and the Brazil basins (P- 333 value= 0.019), and between the Pacific and the Canary basins (P-value: 0.011), suggesting a 334 higher abundance of this bacterium in the Atlantic Ocean. Despite finding these differences 335 between oceans, we did not find significant differences between plankton size fractions, 336 indicating that the isolate could be present in both the free-living (0.2-0.8 µm) and particle- 337 attached (0.8-20 µm) bacterial communities (Figure 3). Moreover, by using bathypelagic 338 metagenomes a Metagenome Assembled Genome (MAG) was reconstructed, the MAG-0289, 339 which genome aligned at 99 % with the Alteromonas sp. ISS312 strain genome and it has a 340 99.34 % of average nucleotide identity (ANI). The FRA of MAG-0289 and ISS312 strain 341 against the bathypelagic metagenomes displayed identical biogeographical patterns 342 (Supplementary Figure S5), indicating that the MAG-0289 is a good representative of the 343 ISS312 strain. 344 345 Our results confirm the prevalence and wide distribution for Alteromonas mediterranea species 346 carrying Hg resistance genes across the global bathypelagic ocean but also shows its occurrence 347 in both plankton size fractions analyzed. To the best of our knowledge the detection and 348 characterization of mer genes from non-contaminated open ocean waters at a large scale has not 349 been previously reported. 350 9 351 Recovery of merA and merB homologous gene variants of ISS312 from marine microbial 352 metagenomes and metatranscriptomes 353 354 A remaining question is if merA and merB genes are active under natural ambient Hg 355 concentrations. For answering this question, we have analyzed available metatranscriptomes 356 (metaT) of the Ocean Microbial Reference Gene catalogue V2 (OMRGC.v2)39 from Tara 357 Oceans Expedition through the Ocean Gene Atlas resource38 covering surface, DCM and 358 mesopelagic layers across oceanographic regions. BLASTp search using the merA and merB 359 genes from the ISS312 strain as input allowed us to extract 54 merA and 6 merB homologous 360 (e-value 1E-100) that ranged from 64 to 99.3 % identities in their aminoacids sequences and 361 belonged to gammaproteobacterial lineages mainly from Alteromonadales order and 362 Halomonadaceae family (Supplementary Table S8). These merA and merB transcripts were 363 prevalent across oceans and depths (see Figure 4, Supplementary Figures S6 and S7). We 364 found that merB genes were more transcribed (Figure 4) in the mesopelagic of the North Indian 365 and Eastern South Pacific Oceans, which are well known regions of oxygen minimum zone 366 areas, and also in some stations from surface South Atlantic waters. It is known that MeHg 367 production presents its maximum concentrations usually near the thermocline and in regions 368 with low oxygen concentrations20,71 as those oxygen minimum zone areas, and perhaps bacteria 369 present in those regions encode and transcribe the Hg resistance genes in order to cope with 370 those more elevated MeHg concentrations. 371 372 Outlook 373 374 A combination of culture-dependent analyses with metagenomic and metatranscriptomic 375 analyses from the global photic and aphotic ocean unveiled that biological reductive MeHg 376 degradation capacities were widely distributed and active in the open ocean, especially in the 377 mesopelagic regions. In particular, this study has uncovered that heterotrophic isolates 378 containing mer genes are present in the open ocean from different oceanographic regions, 379 depths but also plankton size fractions. Also, this study reveals that strains closely related 380 phylogenetically presented contrasting minimum inhibitory concentration values, indicating that 381 the level of Hg resistance is strain specific. In particular we show that ISS312 strain isolated 382 from bathypelagic waters of the South Atlantic Ocean presented a strong and fast capacity to 383 degrade MeHg in 24 h. Moreover, the ISS312 strain genome and lineages related harboring 384 merA and merB genes are present and transcribed globally in marine samples across oceans and 385 depths including polar regions. This outcome has important implications in the biogeochemical 386 cycle of Hg as it provides new understanding on the main players driving MeHg levels in the 10 387 ocean and can ultimately help to improve current frameworks for marine food webs and human 388 exposure to MeHg. 389 390 ASSOCIATED CONTENT 391 Supporting Information 392 Supplementary Materials and Methods section including detailed description of some extra 393 analyses and methodologies used in the study. Supplementary Results describe first exploration 394 in the MARINHET collection and phylogenetic analyses with the 16S rRNA genes of all 290 395 tested strains and the merA and merAB phylogenies. Supplementary Figures including 396 description of primers used, extra phylogenetic trees and results from metagenomes and 397 metatranscriptome analyses for detecting presence and activity of merA and merB in natural 398 conditions. Supplementary Tables excel file for a more detailed description of the origin of 399 tested strains, primers construction, PCR screening analyses, physiological characterization of 400 strains and taxonomic classification of detected merA and merB genes in metagenomes and 401 metatranscriptomes of Tara Oceans data. 402 AUTHOR INFORMATION 403 Corresponding Author 404 Olga Sánchez from the Universitat Autònoma de Barcelona (UAB): olga.sanchez@uab.es, tel. 405 (+34) 93 586 8022, FAX (+34) 93 581 2387; and Silvia G. Acinas from the Insitut de Ciències 406 del Mar (ICM-CSIC): sacinas@icm.csic.es, tel. (+34) 93 230 5886, FAX (+34) 93 230 9555. 407 Author Contributions 408 The study was conceptualized by: ISS, SGA, AGB and OS; experimental procedures were 409 performed by: ISS, CPG, LT, MPiF, MC, RCRM-D; data analyses conducted by: ISS, PS, SGA 410 and writing by: ISS, AGB, SGA, OS. * These authors contributed equally. All authors have 411 given approval to the final version of the manuscript. 412 Funding Sources 413 This study was supported by grants: MER_CLUB (863584-MER_CLUB-EMFF-BlueEconomy- 414 2018) from the European Commission to SGA and OS, and Marie Curie Individual Fellowship 415 (H2020-MSCA-IF-2016; project-749645) to AGB. 416 417 ACKNOWLEDGMENT 418 We are grateful to Elisabet Laia Sà for helping in the laboratory. We thank the Spanish ministry 419 of Science, Innovation and Universities for granting ISS with a PhD FPU grant (FPU14/03590). 420 We are also grateful to the MER_CLUB project (863584-MER_CLUB-EMFF-BlueEconomy- 421 2018) for hiring ISS in order to finish this study. 422 11 423 References 424 425 (1) Miller, M. W.; Clarkson, T. W. Mercury, Mercurial, and Mercaptans.; Thomas, 426 Springfield, ILL., 1973. https://doi.org/https://doi.org/10.1016/s0015-6264(75)80173-8. 427 (2) Clarkson, T. W. Mercury: Major Issues in Environmental Health. Environ. Health 428 Perspect. 1993, 100, 31–38. https://doi.org/10.1289/ehp.9310031. 429 (3) UN Environment. Global Mercury Assesment 2018.; UN Environment Programme, 430 Chemicals and Health Branch Geneva, Switzerland, 2019. 431 (4) Eriksen, H. H.; Perrez, F. X. The Minamata Convention: A Comprehensive Response to 432 a Global Problem. Rev. Eur. Comp. Int. Environ. Law 2014, 23 (2), 195–210. 433 https://doi.org/10.1111/reel.12079. 434 (5) Saiz-Lopez, A.; Sitkiewicz, S. P.; Roca-Sanjuán, D.; Oliva-Enrich, J. M.; Dávalos, J. Z.; 435 Notario, R.; Jiskra, M.; Xu, Y.; Wang, F.; Thackray, C. P.; Sunderland, E. M.; Jacob, D. 436 J.; Travnikov, O.; Cuevas, C. A.; Acuña, A. U.; Rivero, D.; Plane, J. M. C.; Kinnison, D. 437 E.; Sonke, J. E. Photoreduction of Gaseous Oxidized Mercury Changes Global 438 Atmospheric Mercury Speciation, Transport and Deposition. Nat. Commun. 2018, 9 (1), 439 4796. https://doi.org/10.1038/s41467-018-07075-3. 440 (6) Enrico, M.; Roux, G. Le; Marusczak, N.; Heimbürger, L.-E.; Claustres, A.; Fu, X.; Sun, 441 R.; Sonke, J. E. Atmospheric Mercury Transfer to Peat Bogs Dominated by Gaseous 442 Elemental Mercury Dry Deposition. Environ. Sci. Technol. 2016, 50 (5), 2405–2412. 443 https://doi.org/10.1021/acs.est.5b06058. 444 (7) Mason, R. P.; Sheu, G.-R. Role of the Ocean in the Global Mercury Cycle. Global 445 Biogeochem. Cycles 2002, 16 (4), 1093. https://doi.org/10.1029/2001GB001440. 446 (8) Lehnherr, I.; St. Louis, V. L.; Hintelmann, H.; Kirk, J. L. Methylation of Inorganic 447 Mercury in Polar Marine Waters. Nat. Geosci. 2011, 4 (5), 298–302. 448 https://doi.org/10.1038/ngeo1134. 449 (9) Munson, K. M.; Lamborg, C. H.; Boiteau, R. M.; Saito, M. A. Dynamic Mercury 450 Methylation and Demethylation in Oligotrophic Marine Water. Biogeosciences 2018, 15, 451 6451–6460. https://doi.org/10.5194/bg-15-6451-2018. 452 (10) Monperrus, M.; Tessier, E.; Amouroux, D.; Leynaert, A.; Huonnic, P.; Donard, O. F. X. 453 Mercury Methylation, Demethylation and Reduction Rates in Coastal and Marine 454 Surface Waters of the Mediterranean Sea. Mar. Chem. 2007, 107 (1), 49–63. 455 https://doi.org/10.1016/j.marchem.2007.01.018. 456 (11) Podar, M.; Gilmour, C. C.; Brandt, C. C.; Soren, A.; Brown, S. D.; Crable, B. R.; 457 Palumbo, A. V.; Somenahally, A. C.; Elias, D. A. Global Prevalence and Distribution of 458 Genes and Microorganisms Involved in Mercury Methylation. Sci. Adv. 2015, 1 (9), 459 e1500675. https://doi.org/10.1126/sciadv.1500675. 460 (12) Gionfriddo, C. M.; Tate, M. T.; Wick, R. R.; Schultz, M. B.; Zemla, A.; Thelen, M. P.; 461 Schofield, R.; Krabbenhoft, D. P.; Holt, K. E.; Moreau, J. W. Microbial Mercury 462 Methylation in Antarctic Sea Ice. Nat. Microbiol. 2016, 1 (10), 16127. 463 https://doi.org/10.1038/nmicrobiol.2016.127. 464 (13) Mason, R. P.; Choi, A. L.; Fitzgerald, W. F.; Hammerschmidt, C. R.; Lamborg, C. H.; 465 Soerensen, A. L.; Sunderland, E. M. Mercury Biogeochemical Cycling in the Ocean and 466 Policy Implications. Environ. Res. 2012, 119, 101–117. 467 https://doi.org/10.1016/j.envres.2012.03.013. 468 (14) Harding, G.; Dalziel, J.; Vass, P. Bioaccumulation of Methylmercury within the Marine 469 Food Web of the Outer Bay of Fundy, Gulf of Maine. PLoS One 2018, 13 (7), e0197220. 470 https://doi.org/10.1371/journal.pone.0197220. 471 (15) Mergler, D.; Anderson, H. A.; Chan, L. H. M.; Mahaffey, K. R.; Murray, M.; Sakamoto, 472 M.; Stern, A. H. Methylmercury Exposure and Health Effects in Humans: A Worldwide 473 Concern. Ambio 2007, 36 (1), 3–11. https://doi.org/10.1579/0044- 474 7447(2007)36[3:MEAHEI]2.0.CO;2. 475 (16) Karagas, M. R.; Choi, A. L.; Oken, E.; Horvat, M.; Schoeny, R.; Kamai, E.; Cowell, W.; 476 Grandjean, P.; Korrick, S. Evidence on the Human Health Effects of Low-Level 12 477 Methylmercury Exposure. Environ. Health Perspect. 2012, 120 (6), 799–806. 478 https://doi.org/10.1289/ehp.1104494. 479 (17) Cossa, D.; Averty, B.; Pirrone, N. The Origin of Methylmercury in Open Mediterranean 480 Waters. Limnol. Oceanogr. 2009, 54 (3), 837–844. 481 https://doi.org/10.4319/lo.2009.54.3.0837. 482 (18) Sunderland, E. M.; Krabbenhoft, D. P.; Moreau, J. W.; Strode, S. A.; Landing, W. M. 483 Mercury Sources, Distribution, and Bioavailability in the North Pacific Ocean: Insights 484 from Data and Models. Global Biogeochem. Cycles 2009, 23 (2), GB2010. 485 https://doi.org/10.1029/2008GB003425. 486 (19) Blum, J. D.; Popp, B. N.; Drazen, J. C.; Anela Choy, C.; Johnson, M. W. Methylmercury 487 Production below the Mixed Layer in the North Pacific Ocean. Nat. Geosci. 2013, 6 488 (10), 879–884. https://doi.org/10.1038/ngeo1918. 489 (20) Hammerschmidt, C. R.; Bowman, K. L. Vertical Methylmercury Distribution in the 490 Subtropical North Pacific Ocean. Mar. Chem. 2012, 132–133, 77–82. 491 https://doi.org/10.1016/j.marchem.2012.02.005. 492 (21) Malcolm, E. G.; Schaefer, J. K.; Ekstrom, E. B.; Tuit, C. B.; Jayakumar, A.; Park, H.; 493 Ward, B. B.; Morel, F. M. M. Mercury Methylation in Oxygen Deficient Zones of the 494 Oceans: No Evidence for the Predominance of Anaerobes. Mar. Chem. 2010, 122 (1–4), 495 11–19. https://doi.org/10.1016/J.MARCHEM.2010.08.004. 496 (22) Lamborg, C. H.; Hammerschmidt, C. R.; Bowman, K. L. An Examination of the Role of 497 Particles in Oceanic Mercury Cycling. Philos. Trans. R. Soc. A 2016, 374 (2081), 498 20150297. https://doi.org/10.1098/rsta.2015.0297. 499 (23) Zhang., T.; Hsu-Kim, H. Photolytic Degradation of Methylmercury Enhanced by 500 Binding to Natural Organic Ligands Tong. Nat. Geosci. 2010, 3 (7), 473–476. 501 https://doi.org/10.1038/ngeo892. 502 (24) Seller, P.; Kelly, C. A.; Rudd, J. W. M.; Mac Hutchon, A. R. Photodegradation of 503 Methylmercury in Lakes. Nature 1996, 380 (6576), 694–697. 504 https://doi.org/10.1038/380694a0. 505 (25) Costa, M.; Liss, P. S. Photoreduction of Mercury in Sea Water and Its Possible 506 Implications for Hg0 Air-Sea Fluxes. Mar. Chem. 1999, 68 (1–2), 87–95. 507 https://doi.org/10.1016/S0304-4203(99)00067-5. 508 (26) Stal, L. J.; Cretoiu, M. S. What Is so Special about Marine Microorganisms? 509 Introduction to the Marine Microbiome - from Diversity to Biotechnological 510 Applications. In The Marine Microbiome; Stal, L. J., Cretoiu, M. S., Eds.; Springer, 511 Switzerland, 2016; pp 3–20. https://doi.org/10.1007/978-3-319-33000-6. 512 (27) Wang, F.; Macdonald, R. W.; Armstrong, D. A.; Stern, G. A. Total and Methylated 513 Mercury in the Beaufort Sea: The Role of Local and Recent Organic Remineralization. 514 Environmetal Sci. Technol. 2012, 46, 11821–11828. https://doi.org/10.1021/es302882d. 515 (28) Heimbürger, L.-E.; Cossa, D.; Marty, J.-C.; Migon, C.; Averty, B.; Dufour, A.; Ras, J. 516 Methyl Mercury Distributions in Relation to the Presence of Nano- and 517 Picophytoplankton in an Oceanic Water Column (Ligurian Sea, North-Western 518 Mediterranean). Geochim. Cosmochim. Acta 2010, 74 (19), 5549–5559. 519 https://doi.org/10.1016/J.GCA.2010.06.036. 520 (29) Gworek, B.; Bemowska-Kałabun, O.; Kijeńska, M.; Wrzosek-Jakubowska, J. Mercury in 521 Marine and Oceanic Waters—a Review. Water, Air, Soil Pollut. 2016, 227 (10), 371. 522 https://doi.org/10.1007/s11270-016-3060-3. 523 (30) Barkay, T.; Miller, S. M.; Summers, A. O. Bacterial Mercury Resistance from Atoms to 524 Ecosystems. FEMS Microbiol. Rev. 2003, 27 (2–3), 355–384. 525 https://doi.org/10.1016/S0168-6445(03)00046-9. 526 (31) Boyd, E. S.; Barkay, T. The Mercury Resistance Operon: From an Origin in a 527 Geothermal Environment to an Efficient Detoxification Machine. Front. Microbiol. 528 2012, 3 (October), 349. https://doi.org/10.3389/fmicb.2012.00349. 529 (32) Oremland, R. S.; Miller, L. G.; Dowdle, P.; Connell, T.; Barkay, T. Methylmercury 530 Oxidative Degradation Potentials in Contaminated and Pristine Sediments of the Carson 531 River, Nevada. Appl. Environ. Microbiol. 1995, 61 (7), 2745–2753. 13 532 https://doi.org/10.1128/AEM.61.7.2745-2753.1995. 533 (33) Oremland, R. S.; Culbertson, C. W.; Winfrey, M. R. Methylmercury Decomposition in 534 Sediments and Bacterial Cultures: Involvement of Methanogens and Sulfate Reducers in 535 Oxidative Demethylation. Appl. Environ. Microbiol. 1991, 57 (1), 130–137. 536 https://doi.org/10.1128/AEM.57.1.130-137.1991. 537 (34) Barkay, T.; Gu, B. Demethylation─The Other Side of the Mercury Methylation Coin: A 538 Critical Review. ACS Environ. Au 2021. https://doi.org/10.1021/acsenvironau.1c00022. 539 (35) Oremland, R. S.; Culbertson, C. W.; Winfrey, M. R. Methylmercury Decomposition in 540 Sediments and Bacterial Cultures: Involvement of Methanogens and Sulfate Reducers in 541 Oxidative Demethylation. Appl. Environ. Microbiol. 1991, 57 (1), 130–137. 542 https://doi.org/10.1128/AEM.57.1.130-137.1991. 543 (36) Bowman, K. L.; Collins, R. E.; Agather, A. M.; Lamborg, C. H.; Hammerschmidt, C. R.; 544 Kaul, D.; Dupont, C. L.; Christensen, G. A.; Elias, D. A. Distribution of Mercury- 545 cycling Genes in the Arctic and Equatorial Pacific Oceans and Their Relationship to 546 Mercury Speciation. Limnol. Oceanogr. 2019, 65 (S1), S310–S320. 547 https://doi.org/10.1002/lno.11310. 548 (37) Sanz-Sáez, I.; Salazar, G.; Sánchez, P.; Lara, E.; Royo-Llonch, M.; Sà, E. L.; Lucena, T.; 549 Pujalte, M. J.; Vaqué, D.; Duarte, C. M.; Gasol, J. M.; Pedrós-Alió, C.; Sánchez, O.; 550 Acinas, S. G. Diversity and Distribution of Marine Heterotrophic Bacteria from a Large 551 Culture Collection. BMC Microbiol. 2020, 20 (1), 207. https://doi.org/10.1186/s12866- 552 020-01884-7. 553 (38) Villar, E.; Vannier, T.; Vernette, C.; Lescot, M.; Cuenca, M.; Alexandre, A.; Bachelerie, 554 P.; Rosnet, T.; Pelletier, E.; Sunagawa, S.; Hingamp, P. The Ocean Gene Atlas: 555 Exploring the Biogeography of Plankton Genes Online. Nucleic Acids Res. 2018, 46 556 (W1), W289–W295. https://doi.org/10.1093/nar/gky376. 557 (39) Salazar, G.; Paoli, L.; Alberti, A.; Huerta-Cepas, J.; Ruscheweyh, H. J.; Cuenca, M.; 558 Field, C. M.; Coelho, L. P.; Cruaud, C.; Engelen, S.; Gregory, A. C.; Labadie, K.; 559 Marec, C.; Pelletier, E.; Royo-Llonch, M.; Roux, S.; Sánchez, P.; Uehara, H.; Zayed, A. 560 A.; Zeller, G.; Carmichael, M.; Dimier, C.; Ferland, J.; Kandels, S.; Picheral, M.; 561 Pisarev, S.; Poulain, J.; Acinas, S. G.; Babin, M.; Bork, P.; Boss, E.; Bowler, C.; 562 Cochrane, G.; de Vargas, C.; Follows, M.; Gorsky, G.; Grimsley, N.; Guidi, L.; 563 Hingamp, P.; Iudicone, D.; Jaillon, O.; Kandels-Lewis, S.; Karp-Boss, L.; Karsenti, E.; 564 Not, F.; Ogata, H.; Pesant, S.; Poulton, N.; Raes, J.; Sardet, C.; Speich, S.; Stemmann, 565 L.; Sullivan, M. B.; Sunagawa, S.; Wincker, P. Gene Expression Changes and 566 Community Turnover Differentially Shape the Global Ocean Metatranscriptome. Cell 567 2019, 179 (5), 1068-1083.e21. https://doi.org/10.1016/j.cell.2019.10.014. 568 (40) Singer, E.; Webb, E. A.; Nelson, W. C.; Heidelberg, J. F.; Ivanova, N.; Pati, A.; 569 Edwards, K. J. Genomic Potential of Marinobacter Aquaeolei, a Biogeochemical 570 “Opportunitroph.” Appl. Environ. Microbiol. 2011, 77 (8), 2763–2771. 571 https://doi.org/10.1128/AEM.01866-10. 572 (41) López-Pérez, M.; Gonzaga, A.; Martin-Cuadrado, A.-B.; Onyshchenko, O.; Ghavidel, 573 A.; Ghai, R.; Rodriguez-Valera, F. Genomes of Surface Isolates of Alteromonas 574 Macleodii: The Life of a Widespread Marine Opportunistic Copiotroph. Sci. Rep. 2012, 575 2, 696. https://doi.org/10.1038/srep00696. 576 (42) Handley, K. M.; Lloyd, J. R. Biogeochemical Implications of the Ubiquitous 577 Colonization of Marine Habitats and Redox Gradients by Marinobacter Species. Front. 578 Microbiol. 2013, 4, 136. https://doi.org/10.3389/fmicb.2013.00136. 579 (43) Fontanez, K. M.; Eppley, J. M.; Samo, T. J.; Karl, D. M.; DeLong, E. F. Microbial 580 Community Structure and Function on Sinking Particles in the North Pacific Subtropical 581 Gyre. Front. Microbiol. 2015, 6, 469. https://doi.org/10.3389/fmicb.2015.00469. 582 (44) Ivars-Martinez, E.; Martin-Cuadrado, A.-B.; D’Auria, G.; Mira, A.; Ferriera, S.; 583 Johnson, J.; Friedman, R.; Rodriguez-Valera, F. Comparative Genomics of Two 584 Ecotypes of the Marine Planktonic Copiotroph Alteromonas Macleodii Suggests 585 Alternative Lifestyles Associated with Different Kinds of Particulate Organic Matter. 586 ISME J. 2008, 2 (12), 1194–1212. https://doi.org/10.1038/ismej.2008.74. 14 587 (45) Baumann, L.; Baumann, P.; Mandel, M.; Allen, R. D. Taxonomy of Aerobic Marine 588 Eubacteria. J. Bacteriol. 1972, 110 (1), 402–429. 589 (46) Eilers, H.; Pernthaler, J.; Glöckner, F. O.; Amann, R. Culturability and in Situ 590 Abundance of Pelagic Bacteria from the North Sea. Appl. Environ. Microbiol. 2000, 66 591 (7), 3044–3051. https://doi.org/10.1128/AEM.66.7.3044-3051.2000. 592 (47) Floyd, M. M.; Tang, J.; Kane, M.; Emerson, D. Captured Diversity in a Culture 593 Collection: Case Study of the Geographic and Habitat Distributions of Environmental 594 Isolates Held at the American Type Culture Collection. Appl. Environ. Microbiol. 2005, 595 71 (6), 2813–2823. https://doi.org/10.1128/AEM.71.6.2813-2823.2005. 596 (48) Gärtner, A.; Blümel, M.; Wiese, J.; Imhoff, J. F. Isolation and Characterisation of 597 Bacteria from the Eastern Mediterranean Deep Sea. Antonie van Leeuwenhoek, Int. J. 598 Gen. Mol. Microbiol. 2011, 100, 421–435. https://doi.org/10.1007/s10482-011-9599-5. 599 (49) Lekunberri, I.; Gasol, J. M.; Acinas, S. G.; Gómez-Consarnau, L.; Crespo, B. G.; 600 Casamayor, E. O.; Massana, R.; Pedrós-Alió, C.; Pinhassi, J. The Phylogenetic and 601 Ecological Context of Cultured and Whole Genome-Sequenced Planktonic Bacteria 602 from the Coastal NW Mediterranean Sea. Syst. Appl. Microbiol. 2014, 37 (3), 216–228. 603 https://doi.org/10.1016/j.syapm.2013.11.005. 604 (50) Kai, W.; Peisheng, Y.; Rui, M.; Wenwen, J.; Zongze, S. Diversity of Culturable Bacteria 605 in Deep-Sea Water from the South Atlantic Ocean. Bioengineered 2017, 8 (5), 572–584. 606 https://doi.org/10.1080/21655979.2017.1284711. 607 (51) Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, 608 S.; Cooper, S.; Markowitz, S.; Duran, C.; Thierer, T.; Ashton, B.; Meinties, P.; 609 Drummond, A. Geneious Basic: An Integrated and Extendable Desktop Software 610 Platform for the Organization and Analysis of Sequence Data. Bioinformatics 2012, 28 611 (12), 1647–1649. 612 (52) Wiegand, I.; Hilpert, K.; Hancock, R. E. W. Agar and Broth Dilution Methods to 613 Determine the Minimal Inhibitory Concentration (MIC) of Antimicrobial Substances. 614 Nat. Protoc. 2008, 3 (2), 163–175. https://doi.org/10.1038/nprot.2007.521. 615 (53) Zeder, M.; Ellrott, A.; Amann, R. Automated Sample Area Definition for High- 616 Throughput Microscopy. Cytom. Part A 2011, 79A (4), 306–310. 617 https://doi.org/10.1002/cyto.a.21034. 618 (54) Zeder, M.; Pernthaler, J. Multispot Live-Image Autofocusing for High-Throughput 619 Microscopy of Fluorescently Stained Bacteria. Cytom. Part A 2009, 75A (9), 781–788. 620 https://doi.org/10.1002/cyto.a.20770. 621 (55) Sprouffske, K.; Wagner, A. Growthcurver: An R Package for Obtaining Interpretable 622 Metrics from Microbial Growth Curves. BMC Bioinformatics 2016, 17 (1), 172. 623 https://doi.org/10.1186/s12859-016-1016-7. 624 (56) Hall, B. G.; Acar, H.; Nandipati, A.; Barlow, M. Growth Rates Made Easy. Mol. Biol. 625 Evol 2014, No. 31, 232–238. https://doi.org/doi: 10.1093/molbev/mst187. 626 (57) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic Local 627 Alignment Search Tool. J. Mol. Biol. 1990, 215 (3), 403–410. 628 https://doi.org/10.1016/S0022-2836(05)80360-2. 629 (58) Kozlov, A. M.; Darriba, D.; Flouri, T.; Morel, B.; Stamatakis, A. RAxML-NG: A Fast, 630 Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic Inference. 631 Bioinformatics 2019, 35 (21), 4453–4455. https://doi.org/10.1093/bioinformatics/btz305. 632 (59) Letunic, I.; Bork, P. Interactive Tree Of Life (ITOL) v4: Recent Updates and New 633 Developments. Nucleic Acids Res. 2019, 47 (W1), W256–W259. 634 https://doi.org/10.1093/nar/gkz239. 635 (60) Acinas, S. G.; Sánchez, P.; Salazar, G.; Cornejo-Castillo, F. M.; Sebastián, M.; Logares, 636 R.; Royo-Llonch, M.; Paoli, L.; Sunagawa, S.; Hingamp, P.; Ogata, H.; Lima-Mendez, 637 G.; Roux, S.; González, J. M.; Arrieta, J. M.; Alam, I. S.; Kamau, A.; Bowler, C.; Raes, 638 J.; Pesant, S.; Bork, P.; Agustí, S.; Gojobori, T.; Vaqué, D.; Sullivan, M. B.; Pedrós- 639 Alió, C.; Massana, R.; Duarte, C. M.; Gasol, J. M. Deep Ocean Metagenomes Provide 640 Insight into the Metabolic Architecture of Bathypelagic Microbial Communities. 641 Commun. Biol. 2021, 4 (1), 604. https://doi.org/10.1038/s42003-021-02112-2. 15 642 (61) Duarte, C. M. Seafaring in the 21St Century: The Malaspina 2010 Circumnavigation 643 Expedition. Limnol. Oceanogr. Bull. 2015, 24 (1), 11–14. 644 https://doi.org/10.1002/lob.10008. 645 (62) R core team. R Core Team. A Language and Environment for Statistical Computing. R 646 Foundation for Statistical Computing, Vienna, Austria Https://Www.R-Project. Org/. R 647 Foundation for Statistical Computing: Vienna, Austria 2017. 648 (63) Chiu, H.-H.; Shieh, W. Y.; Lin, S. Y.; Tseng, C.-M.; Chiang, P.-W.; Wagner-Dö Bler, I. 649 Alteromonas Tagae Sp. Nov. and Alteromonas Simiduii Sp. Nov., Mercury-Resistant 650 Bacteria Isolated from a Taiwanese Estuary. Int. J. Syst. Evol. Microbiol. 2007, 57, 651 1209–1216. https://doi.org/10.1099/ijs.0.64762-0. 652 (64) Math, R. K.; Jin, H. M.; Kim, J. M.; Hahn, Y.; Park, W.; Madsen, E. L.; Jeon, C. O. 653 Comparative Genomics Reveals Adaptation by Alteromonas Sp. SN2 to Marine Tidal- 654 Flat Conditions: Cold Tolerance and Aromatic Hydrocarbon Metabolism. PLoS One 655 2012, 7 (4), e35784. https://doi.org/10.1371/journal.pone.0035784. 656 (65) Morishita, K.; Nakamura, K.; Tuchiya, K.; Nishimura, K.; Iwahara, M.; Yagi, O. 657 Removal of Methylmercury from a Fish Broth by Alteromonas Macledii Isolated from 658 Minamata Bay. Japanese J. Water Treat. Biol. 2006, 42 (2), 45–51. 659 https://doi.org/10.2521/jswtb.42.45. 660 (66) Vetriani, C.; Chew, Y. S.; Miller, S. M.; Yagi, J.; Coombs, J.; Lutz, R. A.; Barkay, T. 661 Mercury Adaptation among Bacteria from a Deep-Sea Hydrothermal Vent. Appl. 662 Environ. Microbiol. 2005, 71 (1), 220–226. https://doi.org/10.1128/AEM.71.1.220- 663 226.2005. 664 (67) De, J.; Ramaiah, N.; Mesquita, A.; Verlekar, X. N. Tolerance to Various Toxicants by 665 Marine Bacteria Highly Resistant to Mercury. Mar. Biotechnol. 2003, 5 (2), 185–193. 666 https://doi.org/10.1007/s10126-002-0061-6. 667 (68) De, J.; Ramaiah, N. Characterization of Marine Bacteria Highly Resistant to Mercury 668 Exhibiting Multiple Resistances to Toxic Chemicals. Ecol. Indic. 2007, 7 (3), 511–520. 669 https://doi.org/10.1016/J.ECOLIND.2006.05.002. 670 (69) Zheng, R.; Wu, S.; Ma, N.; Sun, C. Genetic and Physiological Adaptations of Marine 671 Bacterium Pseudomonas Stutzeri 273 to Mercury Stress. Front. Microbiol. 2018, 9, 682. 672 https://doi.org/10.3389/fmicb.2018.00682. 673 (70) Robinson, J. B.; Tuovinen, O. H. Mechanisms of Microbial Resistance and 674 Detoxification of Mercury and Organomercury Compounds: Physiological, Biochemical, 675 and Genetic Analyses. Microbiol. Rev. 1984, 48 (2), 95–124. 676 (71) Lamborg, C.; Bowman, K.; Hammerschmidt, C.; Gilmour, C.; Munson, K.; Selin, N.; 677 Tseng, C. M. Mercury in the Anthropocene Ocean. Oceanography 2014, 27 (1), 76–87. 678 https://doi.org/10.5670/oceanog.2014.11. 679 16 680 Tables 681 682 Table 1. Summary of the PCR screening results for merA and merAB in Alteromonas and 683 Marinobacter strains. Photic includes surface and deep chlorophyll maximum (DCM) isolates, 684 while aphotic includes bathypelagic isolates. NW Mediterranean: North Western Mediterranean. 685 Positives PCR for Total strains with Nº of tested Genus Layer Ocean merA and/or strains merA merAB merAB Southern Ocean 1 0 Indian Ocean 0 1 127 Photic NW Mediterranean 2 0 Alteromonas 33 (13.5%) North Atlantic Ocean 5 0 South Atlantic Ocean 4 0 117 Aphotic South Atlantic Ocean 18 3 Southern Ocean 4 4 33 Photic North Atlantic Ocean 1 0 South Atlantic Ocean 26 16 Marinobacter 41 (89.1%) NW Mediterranean 9 2 13 Aphotic North Atlantic Ocean 1 0 South Atlantic Ocean 1 0 686 687 Figures 688 Tree scale: 0.1 onas p. onas s nas erom mo . sp lterom ero c. Alt ST23RT ISS 726 as IM324C ISS247 IM324C ISS2 S281 IM324C Alt 5 IM32R on 114 IM32 ST7 626; un as 388; A IM3 ST nc. m on IM43RT IS ro 63 ST 0RT ISS D_1 6 ISS 76R T ISS 444 0; u 24C m RT I T ISS e ST ST 061 ISS2 23 EF T39 RT I ; Alt ro ISS249 39 MR 787 Hg CP T IS 6RT 1 JX826 lte Cl nc 3 IM4 7456 4 R IM SS1 ; u 01 ISS 61 00 JN59 .A 32 S EF 3RT 169 3 2 50 88 S 1 12 a S 70 T 24 ST ne 4 7 245 60 EF5 49 I 67 S 72 rra C Me 76 50 1 ST ; A. S ST S 7 te 57 C 5 7.5 40 Hg SS RT S 17 i 39 100 2 D 30 JX 7 6R aus I 7 ed 8 2.5 20 52 T SS IS 2 m 5 14 10 10 85 44 07 IM3 T tr 86 86 3R T I ; A. 07 ; S 2R ISS alic 0 0 1 2 5 R 3 5 ulf ST T23 485 ISS ito T IS 114 a S 00 RT 97 ba S1 4 S4 cte 94 rs CP 151 T IS 499 T R p. S 151 T T ISS S R 2 ium 151 S23 cter ST7 ST 4C IS c. ba 6RT 3 3 ; u n ISS IM 2 5 1 1168 49 ISS06 8 ST72 KJ5 RT IS F RT S 557 IBSTR 100 ST72RT IS S 498 ISS 635 T ST76RT ISS 13 ST151R 6 54 ST23RT ISS 71 ST 85S_6 ST151RT ISS 508 IM434C ISS312 ST76RT ISS 1165 IBSTRF R ISS 1278 T ISS158 ST67RT 61 ST151 T ISS 12 IM33 RT ISS 496 ST67R 1185 JQ0 4C IS S S227 RT IS 5S_15 ST 32431; ST76 8 3 ST 118 ST 85S_8 unc. M 0 ISS 260 AM 17RT arin 10 T 1 oba 76R ISS _12 ID 944 ISS cter ST 5 S 66 76 RT ID MED 523 83 67 ST 8 11 75 ; 6 ST SS 12 67 M ID E 12 Ma I M D1 C RT SS 12 ED 2 IS rinob 76 T I SS RT C IS S40 ID ME DRT ; unc 06 RT SS 125 7 ac 8 T M D1 ter ID E R I IS S4 7 RT ISS S 11 S ISS 125 6 67 ED 2 ISS . Ma ID 529 2C I T sp 5 ST 67R S3 08 JX ED1 . M 12 C IS 347 76R ISS 253 RT IS IDM ED12 ISS38 50 S1 6 C T IDM ED12C T . hyd ISS 12 2 T IS 118 IDM S 6R rbon 4 1 IS 18 ST6 o. IDME 5 1172 KX4263 ST67RT ISS 126 S3 23 ST72RT ISS 634 5 ISS 561 7 ST76RT ISS 1184 I 99 ST 88 67 S4 7RT ST 67 DRT IS T T ISS roca C 67 R ST S ISS 7RT 76 40; Marin ISS ST S4 ST72RT ST ST76R 390 ST 1266 ST6 rin S348 ob 5 0; M ac 3 obacter ter 4603 LN8 sp.. Genus: Alteromonas sp. Marinobacter sp. PCR results: merA merA + merAB merAB only 689 690 Figure 1. Phylogeny of the 16S rRNA gene of Alteromonas and Marinobacter positive 691 strains for merA and/or merAB genes screening. First inner colored strip indicates genus of 692 the strain. Second colored strip indicates presence or absence of genes based on PCR results. 693 Bars indicate results from the MIC experiments: yellow, HgCl2; purple, MeHg. Tolerance 694 values are in µM. JX52807, Sulfitobacter and IM32RT_ISS194 are outgroups of the tree. The 695 numbers in the nodes represent bootstrap percentages > 75%. Names in bold indicate reference 696 sequences: A. australica, Alteromonas australica; A. mediterranea, Alteromonas mediterranea; 697 unc., uncultured; M. hydrocarbono., Marinobacter hydrocarbonoclasticus. 698 699 Figure 2. Growth effect of MeHg in strain ISS312. (A) Growth kinetics of Alteromonas 700 mediterranea strain ISS312 in Zobell broth containing MeHg (control (0 µM), 1 µM, 2.5 µM 701 and 5 µM). µmax indicates the maximum growth rate for each MeHg concentration. 702 Transmission electron microscopy (TEM) images of the strain growing at 0 µM and at 5 µM are 703 shown in the right side of the plot. Details for preparation and observation of samples for TEM 704 are explained in Supplementary Information. (B) MeHg removal by strain ISS312 during the 705 growth curve experiment at 5 µM. Mean and standard deviation from three replicates samples 706 are shown in both graphs. 19 707 ISS312; Alteromonas mediterranea 50 Read recruitment (x1000) 100 200 Latitude 0 Lifestyle FL PA -50 8.54 Mbp 99.9% completeness -100 0 100 708 Longitude 709 710 Figure 3. World map showing the distribution of Alteromonas mediterranea strain ISS312. 711 Size of the dots indicate number of reads (x1000) and color indicate if the reads were recruited 712 in the free-living (FL, 0.2-0.8 µm) or in the particle-attached (PA, 0.8-20 µm) bacterial 713 communities of the bathypelagic samples. 714 715 716 A) Surface B) DCM C) Mesopelagic merA in metaT 9.612e-10 8.836e-5 Plankton size fraction [0.22-3µm] 717 D) E) F) 718 merB in metaT 6.626e-9 3.598e-6 Plankton size fraction [0.22-3µm] 719 Figure 4. Biogeography of the merA and merB transcripts across oceanic regions and 720 depths from microbial metatranscriptomes (metaT) from Tara Oceans Expedition. 721 (A,B,C). Abundance of the merA transcripts found in the Surface, deep chlorophyll maximum 722 (DCM), and Mesopelagic samples. (D,E,F). Abundance of merB transcripts found in the 723 Surface, DCM and Mesopelagic samples. 724 725 726 727 728 For Table of Contents only 20 729 MARINHET merA and merB functional screening culture collection merA merB Physiological characterization Hg MIC assays MeHg growth curves Presence (metaG) and activity (metaT) Global samples: 290 Alteromonas and Marinobacter spp. 730 731 732 733 734 735 736 737 738 739 740 21