Microbial diversity of hypersaline environments: a metagenomic approach

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: CURRENT OPINION IN MICROBIOLOGY on 2015, available at: https://doi.org/10.1016/j.mib.2015.05.002” 1 Microbial diversity of hypersaline environments: a metagenomic approach 1 2 Antonio Ventosa1, Rafael R. de la Haba1, Cristina Sánchez-Porro1 and R. 3 Thane Papke2 4 5 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University 6 of Sevilla, 41012 Sevilla, Spain 7 2Department of Molecular and Cell Biology, University of Connecticut, 06269 8 Storrs, CT, USA. 9 10 Corresponding author: Ventosa, Antonio ([email protected]) 11 12 Abstract 13 Recent studies based on metagenomics and other molecular techniques have 14 permitted a detailed knowledge of the microbial diversity and metabolic 15 activities of microorganisms in hypersaline environments. The current accepted 16 model of community structure in hypersaline environments is that the square 17 archaeon Haloquadratum waslbyi, the bacteroidete Salinibacter ruber and 18 nanohaloarchaea are predominant members at higher salt concentrations, while 19 more diverse archaeal and bacterial taxa are observed in habitats with 20 intermediate salinities. Additionally, metagenomic studies may provide insight 21 into the isolation and characterization of the principal microbes in these 22 Manuscript Click here to view linked References 2 habitats, such as the recently described gammaproteobacterium Spiribacter 23 salinus. 24 Introduction 25 Hypersaline environments are extreme habitats with limited microbial diversity 26 due to the combined effects of several environmental factors including high salt 27 concentrations, temperatures and pH, and low nutrient and oxygen availability. 28 Most environmental studies were carried out on aquatic habitats, represented 29 by saline lakes and solar salterns designed for the commercial production of 30 salt. However, other habitats for example include saline soils, salted foods and 31 other products, hides, and deep-sea brine pools (e.g., Lakes Thetis and Kryos) 32 [1-4]. 33 Hypersaline water habitats are classified as thalassohaline, if derived from 34 marine origin and have salt ratios similar to those of marine waters, or 35 athalassohaline when salt proportions are different from seawater. 36 Thalassohaline habitats are typified by solar salterns, which are constituted by a 37 series of shallow ponds in which the seawater is evaporated until the salts are 38 precipitated. However, not all salts precipitate at the same concentration, since 39 this depends on their solubility. Thus, salterns are thalassohaline only up to the 40 salinities when the first salts precipitated. Most athalassohaline habitats are 41 terminal lakes containing various salts, and dependent upon on the geology and 42 geographic location their fresh waters traverse [1]. 43 Halophilic microorganisms are classically categorized on the basis of their 44 optimal growth in different salt concentrations. The two main groups are the 45 extreme halophiles (optimal growth above 15 % NaCl) and moderate halophiles 46 3 (optimal growth 3-15 % NaCl). In contrast, slight halophiles are able to grow 47 optimally in media with 1-3 % NaCl [1,5]. Classification is based on data 48 obtained in laboratory media and conditions, but optimal growth may be 49 influenced by factors such as temperature or media composition. Ideally, 50 categorization should be based on the environmental conditions at which they 51 naturally live instead laboratory ones. 52 Cellular life in hypersaline habitats is dominated by prokaryotes (archaea and 53 bacteria), with a few microbial eukaryotes, such as photosynthetic and 54 heterotrophic protists and fungi, and the crustacean Artemia salina [1,5-8]. Also, 55 a large proportion of viruses are found [9,10]. The use of culture independent 56 techniques typically demonstrated that the most abundant species in saturated 57 brines are the square haloarchaeon Haloquadratum walsbyi and Salinibacter 58 ruber though there are notable exceptions [11,12]. Recent in situ analyses of 59 several hypersaline habitats discovered a new archaeal class called the 60 nanohaloarchaea [13*], and also provided insight into the isolation of some 61 abundant previously uncultivated microbes, such as the recently described 62 gammaproteobacterium Spiribacter salinus [14-16*]. 63 In this review we will focus on the impact of recent molecular methodologies 64 used for the determination of the microbial diversity of hypersaline habitats, 65 especially those using metagenomics approaches and the implications of these 66 techniques. 67 Microbial diversity of salterns 68 The Bras del Port saltern, located in Santa Pola, South-Alicante, on the Spanish 69 Mediterranean Sea coast may be the best-studied hypersaline system with 70 4 respect to its microbiology with nearly continuous study for >35 years 71 [Reviewed in 17]. The first metagenomic studies on this saltern were focused on 72 the saturated NaCl ponds and showed that the community was dominated by 73 Haloquadratum walsbyi, which demonstrated tremendous genome plasticity and 74 high gene content variability among close relatives [18,19], features later 75 observed in other haloarchaeal species [20,21] and in Salinibacter ruber [22]. 76 Metagenomic studies from the Santa Pola saltern of two intermediate-salinity 77 ponds with 13 % and 19 % NaCl and two crystallizer ponds with 33 % and 37 % 78 NaCl were recently carried out [14,15*,23] (Table 1). The only phyla shared by 79 the four datasets were Euryarchaeota and Bacteroidetes, with Euryarchaeota 80 dominating as salinity increased. The phylum Bacteroidetes had similar 81 abundances in each metagenomic dataset (around 7-15 %) but generic 82 affiliation was different. Members of the class Gammaproteobacteria were 83 abundant in the 13 and 19 % salinity datasets. The microbial diversity in these 84 intermediate salinity ponds was larger, containing representatives from 7-9 85 different higher taxa (Figure 1). 86 The presence of novel, abundant and previously unsuspected microbial groups 87 was detected, including a group of low-GC Actinobacteria related to freshwater 88 strains, a euryarchaeon named Candidatus Haloredivivus which has the lowest 89 GC content described for any euryarchaeon, a high-GC euryarchaeon and a 90 gammaproteobacterium related to Alkalilimnicola and Nitrococcus [14]. Single-91 cell genome analyses have also indicated that new Bacteroidetes groups, 92 different from Salinibacter, might be abundant in these salterns [24]. 93 5 These studies reaffirmed the dominance of the genus Haloquadratum at the 94 highest salinities, and being abundant (but not a majority) in the 19 % pond. 95 Halorubrum is the second most represented genus at high salinity (23.1% of the 96 16S rRNA gene sequences) and Salinibacter is the next most abundant taxon in 97 Santa Pola datasets with 6.4, 4.7 and 9.1 % of the sequences in 19, 33 and 37 98 % salinity ponds, respectively. However, the genera Haloquadratum, 99 Halorubrum and Salinibacter had very low abundances in 13 % NaCl 100 [14,15*,23]. Interestingly, the community structure of a 21 % salinity pond 101 located in southern Spain on the Atlantic shore was more similar at higher 102 taxonomic levels to the Mediterranean 33 % NaCl, rather than the expected 19 103 % pond (Figure 1). However, differences at genus level were detected between 104 those ponds indicating that community structure is not based exclusively on salt 105 concentrations or geographic proximity [23]. 106 The identification of previously unknown diversity through the use of 107 metagenomics has led to the cultivation of new species and genera. Many 108 Gammaproteobacteria were present in the Santa Pola 13 and 19 % salinity 109 pond datasets and related to Alkalilimnicola ehrlichii MLHE-1 and Nitrococcus 110 mobilis Nb-231. Further, additional analyses suggested that they might 111 represent an abundant group of bacteria in these habitats. In an effort to 112 cultivate these abundant taxa, different oligotrophic media were designed and 113 extensive sampling and culturing was undertaken from ponds located in Santa 114 Pola and Isla Cristina, in southern Spain. Two strains, M19-40 and UAH-SO71 115 were isolated and their complete genomes were sequenced [25]. Comparative 116 analysis of metagenomic datasets with these genomes indicates they are 117 distributed worldwide in intermediate salinity habitats (ca. 10-25 % salts), 118 6 representing the first ecologically defined moderate halophile. Recently, strain 119 M19-40 has been described as a new genus and species as Spiribacter salinus 120 (family Ectothiorhodospiraceae); however, several closely related species or 121 genera within the family Ectothiorhodospiraceae are present and await 122 classification [16*]. 123 Saltern crystallizer ponds have among the highest known concentrations of 124 viruses for aquatic systems, and the Santa Pola saltern was explored in order to 125 gain insight into this important group. EHP-1 (environmental halophage 1) was 126 the first nearly complete genome reconstructed from a crystallizer pond with a 127 salinity of 31 % (CR30). Its G+C content and codon usage was similar to that of 128 Haloquadratum walsbyi, suggesting perhaps this is its natural host [26]. 129 Additional metagenomic analyses of viral DNA showed a high number of single 130 nucleotide polymorphisms (SNP) indicating that the viral community was highly 131 diverse and very different from other environments, but some similarities from 132 high salt concentration ponds were found. In addition, similarities in dinucleotide 133 frequency analysis and G+C content allowed the speculation of their putative 134 hosts: Haloquadratum walsbyii and Salinibacter ruber [27]. Comparable 135 observations were found in metatranscriptomic analyses of CR30 viral 136 communities [28], and in a fosmid metagenome library, where 42 different viral 137 genomes were retrieved and assigned to H. walsbyi, S. ruber and one viral 138 cluster that appeared to prey on the Nanoarchaea [29]. 139 A new development in the ability to assign viruses to hosts from sequence data 140 relies on recovering genomes by single-cell genomics technologies and 141 hybridization of viral DNA from the same samples, previously immobilized on a 142 7 microarray. Using this technique authors pinpointed viruses infecting not only 143 cultivable microorganisms but also non-cultivable, the so-called “microbial dark 144 matter” [30*]. 145 Other remarkable studies related to the microbial diversity on salterns using 146 different molecular techniques have been carried out in Sfax saltern in Tunisia 147 [31,32], Maras saltern in the Peruvian Andes [33], Mallorca saltern, Spain 148 [34,35], and Guerrero Negro saltern, Mexico [12]. 149 Lake Tyrrell 150 Located northwest of Melbourne, Australia, this thalassohaline salt lake 151 experiences large seasonal changes in temperature, oxygen, solar radiation, 152 pH, water levels, and especially in salt concentration [36,37] and provides a 153 nice comparison to model system established in Santa Pola, Spain. The very 154 first metagenomic analyses of this site revealed the exciting discovery of a 155 novel major lineage of halophilic Archaea, for which the creation of a new class 156 Nanohaloarchaea was proposed [13*]. Despite having a common ancestor with 157 the Halobacteriaceae it possessed many unique characteristics including small 158 cell size (0.6 m), reduced genome (1.2 Mbp) with a single rRNA operon, low 159 G+C content, peculiar amino acid composition, absence of gas vesicle genes 160 and atypical carbohydrate metabolism pathways [13*]. Independent analyses 161 using environmental 16S rRNA genes, quantitative FISH, and statistical study of 162 the assembled metagenomic reads estimated that the Nanohaloarchaea 163 comprised 10-25 % of the archaeal community in Lake Tyrrell [13*]. Further 164 studies confirmed that typical haloarchaeal genera (e.g., Haloquadratum, 165 8 Halorubrum, Nanohaloarchaea, Halonotius, Halorhabdus, Halobaculum and 166 Haloarcula) and Salinibacter dominate the lake microbial diversity [13*,38-40]. 167 Lake Tyrell community members are dynamic over time and space, particularly 168 on timescales of months to years [39]. Relative abundances appear to be 169 affected by seasonal fluctuations linked to ionic concentration. Haloquadratum 170 and Nanohaloarchaea-related sequences (low genomic G+C composition) were 171 more abundant in summer than in winter, whereas Halorubrum and Haloarcula-172 related sequences (higher G+C content) followed the opposite trend [39,40]. 173 Elevated magnesium, potassium and sulfate concentrations were positively 174 correlated to Haloquadratum abundance, and negatively to Halorubrum, 175 Haloarcula, Halonotius, Halobaculum and Salinibacter. Nanohaloarchaea and 176 Halorhabdus did not show any correlation [40] suggesting their abundance was 177 independent of Haloquadratum, and competition among them for a similar niche 178 space [39,40]. 179 Eukaryotes had low abundance and richness with the flagellate Colpodella sp. 180 and the green alga Dunaliella salina as the predominant representatives, but 181 they may play important ecological roles [41]. 182 Populations of DNA viruses found in hypersaline systems (infecting bacterial, 183 archaeal or eukaryotic cells), also called halovirus [27], were not well conserved 184 especially across time and space. About 1,000 dominant viral populations may 185 be present at any specific time (an underestimate given RNA viruses were not 186 analysed), but they were stable only for days or a few weeks, and were 187 demonstrated to be dynamic on timescales of months or years [42,43]. Such 188 findings indicate that viral diversity studies based on one or a few samples are 189 15 diversity of an intermediate salinity hypersaline habitat assessed by 330 metagenomics. FEMS Microbiol Ecol 2014. 88:623-635. 331 Analysis of a metagenome obtained by pyrosequencing of the total prokaryotic 332 DNA from a pond with intermediate salinity (13 % salts) from a saltern in Santa 333 Pola, Spain, showed a large microbial diversity, representing seven major 334 higher taxa, being most sequences (57 %) not assigned to any previously 335 described taxon. 336 * 16. León MJ, Fernández AB, Ghai R, Sánchez-Porro C, Rodriguez-Valera F, 337 Ventosa A: From metagenomics to pure culture: isolation and 338 characterization of the moderately halophilic bacterium Spiribacter 339 salinus gen. nov., sp. nov. Appl Environ Microbiol 2014, 80:3850-3857. 340 Based on the previous metagenomic analyses, this paper describes the 341 isolation, taxonomic characterization andfeatures of a new 342 gammaproteobacterium, designated as Spiribacter salinus, abundant in 343 hypersaline environments with intermediate salinities. 344 17. Ventosa A, Fernández AB, León MJ, Sánchez-Porro C, Rodriguez-Valera F: 345 The Santa Pola saltern as a model for studying the microbiota of 346 hypersaline environments. Extremophiles 2014, 18:811-824. 347 18. Legault BA, Lopez-Lopez A, Alba-Casado JC, Doolittle WF, Bolhuis H, 348 Rodriguez-Valera F, Papke RT. Environmental genomics of "Haloquadratum 349 walsbyi" in a saltern crystallizer indicates a large pool of accessory genes 350 in an otherwise coherent species. BMC Genomics 2006, 7:171. 351 16 19. Cuadros-Orellana S, Martin-Cuadrado AB, Legault B, D'Auria G, 352 Zhaxybayeva O, Papke RT, Rodriguez-Valera F: Genomic plasticity in 353 prokaryotes: the case of the square haloarchaeon. ISME J 2007, 1:235-245. 354 20. Fullmer MS, Soucy SM, Swithers KS, Makkay AM, Wheeler R, Ventosa A, 355 Gogarten JP, Papke RT: Population and genomic analysis of the genus 356 Halorubrum. Front Microbiol 2014, 5:140. 357 21. Ram Mohan N, Fullmer MS, Makkay AM, Wheeler R, Ventosa A, Naor A, 358 Gogarten JP, Papke RT: Evidence from phylogenetic and genome 359 fingerprinting analyses suggests rapidly changing variation in 360 Halorubrum and Haloarcula populations. Front Microbiol 2014, 5:143. 361 22. Pasic L, Rodriguez-Mueller B, Martin-Cuadrado AB, Mira A, Rohwer F, 362 Rodriguez-Valera F: Metagenomic islands of hyperhalophiles: the case of 363 Salinibacter ruber. BMC Genomics 2009, 10:570. 364 23. Fernández AB, Vera-Gargallo B, Sánchez-Porro C, Ghai R, Papke RT, 365 Rodriguez-Valera F, Ventosa A: Comparison of prokaryotic community 366 structure from Mediterranean and Atlantic saltern concentrator ponds by 367 a metagenomic approach. Front Microbiol 2014, 5:196. 368 24. Gomariz M, Martínez-García M, Santos F, Rodriguez F, Capella-Gutiérrez 369 S, Gabaldón T, Rosselló-Móra R, Meseguer I, Antón J: From community 370 approaches to single-cell genomics: the discovery of ubiquitous 371 hyperhalophilic Bacteroidetes generalists. ISME J 2015, 9:16-31. 372 25. López-Pérez M, Ghai R, Leon MJ, Rodríguez-Olmos A, Copa-Patiño JL, 373 Soliveri J, Sanchez-Porro C, Ventosa A, Rodriguez-Valera F: Genomes of 374 17 "Spiribacter", a streamlined, successful halophilic bacterium. BMC 375 Genomics 2013, 14:787. 376 26. Santos F, Meyerdierks A, Peña A, Rosselló-Mora R, Amann R, Antón J: 377 Metagenomic approach to the study of halophages: the environmental 378 halophage 1. Environ Microbiol 2007, 9:1711-1723. 379 27. Santos F, Yarza P, Parro V, Briones C, Antón J: The metavirome of a 380 hypersaline environment. Environ Microbiol 2010, 12:2965-2976. 381 28. Santos F, Moreno-Paz M, Meseguer I, López C, Rosselló-Mora R, Parro V, 382 Antón J: Metatranscriptomic analysis of extremely halophilic viral 383 communities. ISME J 2011, 5:1621-1633. 384 29. Garcia-Heredia I, Martin-Cuadrado AB, Mojica FJ, Santos F, Mira A, Antón 385 J, Rodriguez-Valera F: Reconstructing viral genomes from the environment 386 using fosmid clones: the case of haloviruses. PLoS One 2012, 7:e33802. 387 * 30. Martínez-García M, Santos F, Moreno-Paz M, Parro V, Antón J: Unveiling 388 viral-host interactions within the 'microbial dark matter'. Nat Commun 389 2014, 5: 4542. 390 This study described a way, based on single-cell genomic technologies, to 391 assign viruses to hosts that does not rely on previous information about either of 392 them nor requires their cultivation. It was used to pinpoint viruses infecting 393 Nanohaloarchaeota. 394 31. Boujelben I, Gomariz M, Martínez-García M, Santos F, Peña A, López C, 395 Antón J, Maalej S: Spatial and seasonal prokaryotic community dynamics 396 18 in ponds of increasing salinity of Sfax solar saltern in Tunisia. Antonie van 397 Leeuwenhoek 2012, 101:845-857. 398 32. Boujelben I, Yarza P, Almansa C, Villamor J, Maalej S, Antón J, Santos F: 399 Virioplankton community structure in Tunisian solar salterns. Appl Environ 400 Microbiol 2012, 78:7429-7437. 401 33. Maturrano L, Santos F, Rosselló-Mora R, Antón J: Microbial diversity in 402 Maras salterns, a hypersaline environment in the Peruvian Andes. Appl 403 Environ Microbiol 2006, 72:3887-3895. 404 34. Munoz R, López-López A, Urdiain M, Moore ER, Rosselló-Móra R: 405 Evaluation of matrix-assisted laser desorption ionization-time of flight 406 whole cell profiles for assessing the cultivable diversity of aerobic and 407 moderately halophilic prokaryotes thriving in solar saltern sediments. Syst 408 Appl Microbiol 2011, 34:69-75. 409 35. López-López A, Richter M, Peña A, Tamames J, Rosselló-Móra R: New 410 insights into the archaeal diversity of a hypersaline microbial mat 411 obtained by a metagenomic approach. Syst Appl Microbiol 2013, 36:205-412 214. 413 36. Macumber PG: Hydrological processes in the Tyrrell Basin, 414 southeastern Australia. Chem Geol 1992, 96:1-18. 415 37. Williams WD: Anthropogenic salinisation of inland waters. Hydrobiology 416 2001, 466:329-337. 417 19 38. Podell S, Ugalde JA, Narasingarao P, Banfield JF, Heidelberg KB, Allen EE: 418 Assembly-driven community genomics of a hypersaline microbial 419 ecosystem. PLoS One 2013, 8:e61692. 420 39. Emerson JB, Andrade K, Thomas BC, Norman A, Allen EE, Heidelberg KB, 421 Banfield JF: Virus-host and CRISPR dynamics in Archaea-dominated 422 hypersaline Lake Tyrrell, Victoria, Australia. Archaea 2013, 2013:370871. 423 40. Podell S, Emerson JB, Jones CM, Ugalde JA, Welch S, Heidelberg KB, 424 Banfield JF, Allen EE: Seasonal fluctuations in ionic concentrations drive 425 microbial succession in a hypersaline lake community. ISME J 2014, 426 8:979-990. 427 41. Heidelberg KB, Nelson WC, Holm JB, Eisenkolb N, Andrade K, Emerson 428 JB: Characterization of eukaryotic microbial diversity in hypersaline Lake 429 Tyrrell, Australia. Front Microbiol 2013, 4:115. 430 42. Emerson JB, Thomas BC, Andrade K, Allen EE, Heidelberg KB, Banfield 431 JF: Dynamic viral populations in hypersaline systems as revealed by 432 metagenomic assembly. Appl Environ Microbiol 2012, 78:6309-6320. 433 43. Emerson JB, Thomas BC, Andrade K, Heidelberg KB, Banfield JF: New 434 approaches indicate constant viral diversity despite shifts in assemblage 435 structure in an Australian hypersaline lake. Appl Environ Microbiol 2013, 436 79:6755-6764. 437 44. Bowman JP, McCammon SA, Rea SM, McMeekin TA: The microbial 438 composition of three limnologically disparate hypersaline Antarctic lakes. 439 FEMS Microbiol Lett 2000, 183:81-88. 440 20 45. Yau S, Lauro FM, DeMaere MZ, Brown MV, Thomas T, Raftery MJ, 441 Andrews-Pfannkoch C, Lewis M, Hoffman JM, Gibson JA, Cavicchioli R: 442 Virophage control of antarctic algal host-virus dynamics. Proc Natl Acad 443 Sci U S A 2011, 108:6163-6168. 444 46. Yau S, Lauro FM, Williams TJ, Demaere MZ, Brown MV, Rich J, Gibson JA, 445 Cavicchioli R: Metagenomic insights into strategies of carbon conservation 446 and unusual sulfur biogeochemistry in a hypersaline Antarctic lake. ISME 447 J 2013, 7:1944-1961. 448 * 47. DeMaere MZ, Williams TJ, Allen MA, Brown MV, Gibson JA, Rich J, Lauro 449 FM, Dyall-Smith M, Davenport KW, Woyke T, Kyrpides NC, Tringe SG, 450 Cavicchioli R: High level of intergenera gene exchange shapes the 451 evolution of haloarchaea in an isolated Antarctic lake. Proc Natl Acad Sci 452 USA 2013, 110:16939-16944. 453 Analysis of metagenomic data and genomes of four haloarchaeal isolates 454 representing different genera revealed that they shared long continuous high-455 identity regions (up to 35 kb) supporting the hypothesis of haloarchaeal 456 population homogenization through intergeneric gene exchange while 457 maintaining sympatric speciation. 458 48. Williams TJ, Allen MA, DeMaere MZ, Kyrpides NC, Tringe SG, Woyke T, 459 Cavicchioli R: Microbial ecology of an Antarctic hypersaline lake: genomic 460 assessment of ecophysiology among dominant haloarchaea. ISME J 2014, 461 8:1645-1658. 462 49. Glatz RE, Lepp PW, Ward BB, Francis CA: Planktonic microbial 463 community composition across steep physical/chemical gradients in 464 21 permanently ice-covered Lake Bonney, Antarctica. Geobiology 2006, 4:53-465 67. 466 50. Mosier AC, Murray AE, Fritsen CH: Microbiota within the perennial ice 467 cover of Lake Vida, Antarctica. FEMS Microbiol Ecol 2007, 59:274-288. 468 51. Matsuzaki M, Kubota K, Satoh T, Kunugi M, Ban S, Imura S: Dimethyl 469 sulfoxide-respiring bacteria in Suribati Ike, a hypersaline lake, in Antarctic 470 and the marine environment. Polar Biosci 2006, 20:73-81. 471 52. Naganuma T, Hua PN, Okamoto T, Ban S, Imura S, Kanda H: Depth 472 distribution of eury-haline halophilic bacteria in Suribati Ike, a meromictic 473 lake in East Antarctica. Polar Biol 2005, 28:964-970. 474 53. Wilkins D, Yau S, Williams TJ, Allen MA, Brown MV, DeMaere MZ, Lauro 475 FM, Cavicchioli R: Key microbial drivers in Antarctic aquatic environments. 476 FEMS Microbiol Rev 2013, 37:303-335. 477 54. Papke RT1, Zhaxybayeva O, Feil EJ, Sommerfeld K, Muise D, Doolittle WF: 478 Searching for species in haloarchaea. Proc Natl Acad Sci USA 479 2007,104:14092-14097. 480 55. Williams D, Gogarten JP, Papke RT: Quantifying homologous 481 replacement of loci between haloarchaeal species. Genome Biol Evol 2012, 482 4:1223-1244. 483 56. La Scola B, Desnues C, Pagnier I, Robert C, Barrassi L, Fournous G, 484 Merchat M, Suzan-Monti M, Forterre P, Koonin E, Raoult D: The virophage as 485 a unique parasite of the giant mimivirus. Nature 2008, 455:100-104. 486 22 57. Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JL, D’Auria G, de Lima 487 Alves F, La Cono V, Genovese M, McKew BA, Hayes SL, et al.: Limits of life 488 in MgCl2-containing environments: chaotropicity defines the window. 489 Environ Microbiol 2007, 9:801–813. 490 58. Bodaker I, Sharon I, Suzuki MT, Feingersch R, Shmoish M, Andreishcheva 491 E, Sogin ML, Rosenberg M, Maguire ME, Belkin S, et al.: Comparative 492 community genomics in the Dead Sea: an increasingly extreme 493 environment. ISME J 2010, 4:399-407. 494 59. Oren A: Microbiological studies in the Dead Sea: future challenges 495 toward the understanding of life at the limit of salt concentrations. 496 Hydrobiologia 1999, 405:1-9. 497 60. Rhodes ME, Fitz-Gibbon ST, Oren A, House CH: Amino acid signatures 498 of salinity on an environmental scale with a focus on the Dead Sea. 499 Environ Microbiol 2010, 12:2613-2623. 500 501 502 23 Legend to figure 503 504 Figure 1. A. Distribution of Bacteria and Archaea in Santa Pola saltern 505 (metagenomic datasets SS13 to SS37), Isla Cristina saltern (metagenomic 506 dataset IC21) and three hypersaline lakes, Lake Tyrrell, Organic Lake and 507 Deep Lake. B. Relative percentages of the different higher taxa in the 508 ponds of Santa Pola saltern (datasets SS13, SS19, SS33 and SS37, 509 corresponding to ponds with 13 %, 19 %, 33 % and 37 % salinity, 510 respectively), and Isla Cristina saltern (dataset IC21, corresponding to a 511 pond with 21 % salinity). Data obtained from references 13*, 14, 15*, 23, 46 512 and 47*. 513 24 Table 1. Summary features of metagenomic datasets from Spanish solar salterns, Lake Tyrrell (Australia) and hypersaline Antarctic lakes. NA, data not available. Datasets Number of reads Dataset size Average read length Accession number Reference Santa Pola Saltern (SS13) 1,443,593 441 Mb 305 bp SRP028290 [15*] Santa Pola Saltern (SS19) 1,315,302 475 Mb 361 bp SRP007685 [14] Santa Pola Saltern (SS33) 842,872 309 Mb 367 bp SRP028290 [23] Santa Pola Saltern (SS37) 760,740 309 Mb 417 bp SRP007685 [14] Isla Cristina saltern (IC21) 1,223,923 486 Mb 397 bp SRP029970 [23] Lake Tyrrell (0.3 m depth) 11,159,862 4559 Mb 409 bp PRJNA59457a [13*,38,40] Lake Tyrrell (metavirome) 56,142,238 6417 Mb 114 bp PRJNA81851a [42] Organic Lake (Antarctica) 6,016,538 2400 Mb 399 bp NA [46] Deep Lake (Antarctica) 53,659,088 ~10000 Mb 186 bp Gs0000582, Gs0000585, Gs0000586, Gs0000587, Gs0000592, Gs0000594, Gs0000595 [47*] a Bioproject accession number corresponding to a set of different samples.