Microbiome of the Black Sea water column analyzed by genome centric metagenomics

Background The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former albeit with lower salinity and (mostly) temperature. In spite of its well-known hydrology and physico chemistry, this enormous water mass remains poorly studied at the microbial genomics level. Results We have sampled its different water masses and analyzed the microbiome by classic and genome-resolved metagenomics generating a large number of metagenome-assembled genomes (MAGs) from them. The oxic zone presents many similarities to the global ocean while the euxinic water mass has similarities to other similar aquatic environments of marine or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environments and includes Cyanobacteria (Synechococcus), photoheterotrophs (largely with marine relatives), facultative/microaerophilic microbes again largely marine, chemolithotrophs (N and S oxidizers) and a large number of anaerobes, mostly sulfate reducers but also a few methanogens and a large number of “dark matter” streamlined genomes of largely unpredictable ecology. Conclusions The Black Sea presents a mixture of similarities to other water bodies. The photic zone has many microbes in common with that of the Mediterranean with the relevant exception of the absence of Prochlorococcus. The chemocline already presents very different characteristics with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. Finally the euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy generating metabolism and a small but detectable methanogenesis.We are adding critical information about this unique and important ecosystem and its microbiome.

genomes (MAGs) from them. The oxic zone presents many similarities to the global ocean 27 while the euxinic water mass has similarities to other similar aquatic environments of marine 28 or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very 29 well the different types of metabolisms expected in this kind of environments and includes 30 Cyanobacteria (Synechococcus), photoheterotrophs (largely with marine relatives), 31 facultative/microaerophilic microbes again largely marine, chemolithotrophs (N and S 32 oxidizers) and a large number of anaerobes, mostly sulfate reducers but also a few methanogens 33 and a large number of "dark matter" streamlined genomes of largely unpredictable ecology. Gammaproteobacteria that co-occurred in these samples were MAGs with similarity to 183 Luminiphilus (11 MAGs) and Litoricola (3 MAGs) genera. Another relevant taxon from 184 marine systems was the former marine group-II Euryarchaeota (Thermoplasmatota according 185 to GTDB taxonomy). We retrieved six genomes affiliating to the family Poseidoniaceae and 186 other six to the genus Poseidonia. Only one genome was obtained affiliating to 187 Thalassoarchaeaceae. Finally, three ultra-small (1 Mb of estimated genome size) 188 Marinimicrobia MAGs were obtained from oxic metagenomes, which so far are classified by 189 the GTDB as genus Marinisoma. 190 191 Black Sea pycnocline MAGS 192 193 The redoxcline of the Black Sea presented the most metabolically diverse set of pathways 194 among all analyzed samples ( Fig. 2A). The main environmental variables that statistically 195 grouped with the pycnocline were total nitrogen (TN) and nitrate, which were clearly 196 associated with the different N cycle pathways that completed its biogeochemical cycle in this 197 layer. The highest abundance of N pathways corresponded with denitrification (nitrogen gas as 198 the final product), nitrate/nitrite ammonification and dissimilatory nitrate reduction (with 199 ammonium as the final product), but the N cycle was also completed with ammonia oxidation 200 and N fixation pathways detected both in total reads and MAGs (see below). Nonetheless, 201 various other metabolisms coexisted in this thin layer where oxygen is extinguished. We 202 noticed the presence of anoxygenic photosynthesis, exemplified by MAG BS150m- G13 203 showing > 99 % of ANI with Chlorobium phaeobacteroides, a green sulfur bacterium (GSB) 204 originally isolated from the Black Sea [18] (GCA_000020545.1), that was undergoing a nearly 205 monoclonal bloom (Fig. S3) We also compared our pycnocline dataset with previously available metagenomes from the 241 redoxcline from Cariaco Basin (Venezuela) [21] (Fig. S5). Overall, it seems that 242 Marinisomatota/Marinimicrobia and Gammaproteobacteria chemolithotrophic groups are the 243 most abundant key players of these two marine redoxclines, accounting for more than 50 % of 244 total microbial biomass (Fig. S5A). However, it must be noted that only a few species retrieved 245 as MAGs from the Black Sea were detected in such a similar habitat (Fig. S5C). Among them, 246 two chemolithotrophic Gammaproteobacterial representatives (Ca. Thioglobus and a novel 247 species BS150m-G30 classified only at the order level as o__GCA-2400775 by GTDB), sulfate 248 reducers (Desulfatiglandales), denitrifying and hydrogen-producing Marinimicrobia (three 249 species) and one Actinobacteria (a novel species from the marine MedAcidi-G1 group). Apart 250 from their metabolic potential fitting with microbial lifestyles from pycnocline layers, these 251 species could play key roles in other marine redoxclines and oxygen minimum zones (OMZs), 252 as their detection in two largely separated biomes with different salinities (ca. 2 % in the Black 253 Sea and 3.5 % in the Cariaco Basin) indicate a widespread distribution in oxygen-depleted 254 marine niches. Planctomycetes, andChloroflexi MAGs as well as in the unassembled reads (being completely 278 absent from oxic datasets), suggesting that these electron transfer complexes are not exclusive 279 of methanogens. As seen by the dbRDA, we also noted a global predominance of mixed-acid 280 fermentation pathways (with ethanol, lactate, acetate, formate or CO2/H2 as products) and 281 hydrogen uptake hydrogenases that couple with sulfate, fumarate, CO2 or nitrate reduction, 282 thus conforming a complex syntrophic network of microbes. This networking of syntrophic 283 microbes (considered here as interspecies H transfer) includes the abovementioned uncultured 284 taxa plus accompanying streamlined members of the "microbial dark matter" such as 285 Omnitrophota, Patescibacteria (Ca. Microgenomates, Portnoybacteria, Paceibacteria) or 286 Nanoarchaeota (Ca. Aenigmarchaeota, Woesearchaeota, Pacearchaeota), groups from which 287 we also obtained MAGs (see Table 2). Various types of hydrogenases and hydrogen 288 metabolism pathways grouped with the 750 m mesopelagic sample in the dbRDA plot (Fig. 2) 289 and were found in the vast majority of microbes inhabiting this sulfide enriched waters, 290 including NAD-reducing bidirectional (hox genes) and uptake hydrogenases (hup genes), NiFe 291 (hyp genes) and FeFe (hym genes) hydrogenases, Coenzyme F420-reducing hydrogenases or 292 carbon monoxide induced hydrogenases (CooHL genes), all of which showed the highest gene/ 293 recA ratios (from 0.2 in hym genes to 1-2 for hyp and hoxF) in euxinic waters. 294

295
It was remarkable the presence of two Actinobacteria MAGs (BS750m-G1/G2) in these 296 sulfide-rich waters. These yet unclassified members have their highest resemblance with 297 presented a small predicted genome size (ca. 1.2 Mb) and was an obligate fermenter (mainly 308 producing ethanol, H2/CO2 and lactate). Another group of streamlined members of the 309 microbial dark matter were Aenigmarchaeota (BS750m-G24/36/81/83/) and Nanoarchaeota 310 (BS750m-G11/13/70) MAGs, which had estimated genome sizes of 1-1.5 Mb. Among their 311 metabolic potential, they were also mixed-acid fermenters, including lactate or H2/CO2 as 312 fermentation by-products, which would fuel the sulfate reducers, conforming a syntrophic 313 network with the rest of mixed-acid fermenters. Finally, another set of microbes of small 314 genome sizes (0.6-1.6 Mb) were Patescibacteria (former Candidate Phyla Radiation). We must 315 highlight the presence of the protein VirB4, associated with type IV secretion systems that 316 work as injectors into host cells [22], in Ca. Microgenomates BS750m-G73/74, Ca. 317 Paceibacteria BS750m-G71/75 and Ca. Portnoybacteria bacterium BS750m-G76. These 318 proteins were unique for these microbes in the entire euxinic waters, which suggests a parasitic 319 lifestyle from which these Patescibacteria could translocate nutrients, proteins, and DNA from 320 or to a putative host [22]. 321 322

Similarities between Black Sea datasets assessed by read and recruitment analysis 323
To assess the representativity of our samples we also compared the reads between our Black As we approach the euxinic mesopelagic and bathypelagic waters, we tend to encounter a 371 fundamental domination of sulfate reduction coupled with a complex variety of syntrophic 372 networks that feed the ecosystem. In this sense, Desulfobacterota is a good example of a 373 syntrophic phylum that could be able to accept electrons from other electron donors, as noted 374 previously in marine sediments [28]. It appears that the extremely high abundance of sulfate-reducers in the Black Sea has displaced methanogens, which are present in the water column 376 but at low numbers, having obtained Bathyarchaeota [29,30] representatives and a single 377 example of a Syntrophoarchaeum from this study. In fact, this last microbe could be performing 378 reverse methanogenesis or anaerobic methane oxidation (ANME) in the system. Recently, 379 members of this newly identified species have shown the complete oxidation of butane during 380 the anaerobic methane oxidation process [31]. However, it appears that the competition 381 between methanogens and sulfate-reducers for acetate is dominated by the latter, which also 382 take a fundamental role in the syntrophic network by uptaking all the H2 produced by the 383 fermenters. Among all of the associated microbiota, we must highlight the presence of 384

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The author(s) declare no competing interest. 504

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All data derived from this work is publicly available in the NCBI-Genbank databases. 506

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