Metagenomes of a Crystallizer Pond from Isla Cristina Saltern in Spain

Metagenomes of a Crystallizer Pond from Isla Cristina Saltern in Spain Alicia García-Roldán,aRafael R. de la Haba,aBlanca Vera-Gargallo,aCristina Sánchez-Porro,aAntonio Ventosaa a Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain ABSTRACT The metagenomic sequences of the prokaryotic microbiota from the brine of a crystallizer pond with 42% (wt/vol) salinity of a saltern located in Isla Cristina, Huelva, southwest Spain, were obtained by Illumina. Haloarchaea and members of the bacterial genus Salinibacter were the most abundant prokaryotes. Solar salterns are multipond systems used for the commercial production of salt by evaporation of salted water, and they are excellent models for the study of the microbial communities living in a wide range of salt concentrations, from seawater to salt saturation (1). One of the salterns that has been extensively studied is Santa Pola saltern, located on the Mediterranean coast in Spain (2). In contrast, Atlantic salterns have received less attention. Isla Cristina saltern has been revealed as a source of interesting new prokaryotic microorganisms (3–12). In 2014, we reported early studies based on a metagenome obtained by pyrosequencing (Roche 454 GS-FLX technology) from the brine of an intermediate pond with 21% (wt/vol) salts (13). The analysis of this metagenome (designated IC21) indicated that Euryarchaeota (84%), mainly represented by the haloarchaeal genera Halorubrum (65.8%), Natronomonas (3.2%), and Haloquadratum (1.9%), was the most abundant phylum, followed by Bacteroidota (8.0%), dominated by the genus Psychroflexus (4.6%), and Gammaproteobacteria (7.0%), with the genus Spiribacter (1.1%) being the most abundant (14). In this report, we describe the sequencing of two metagenomic databases from the prokaryotic fraction of an Isla Cristina saltern crystallizer pond, in which salts precipitate, with a salinity of 42% (wt/vol). These were designated 20IC42-1 and 20IC42-2 and were obtained by Illumina technology. Specifically, an 8-L brine sample was collected from the surface of a crystallizer pond with 42% (wt/vol) total salts (GPS coordinates 37°12939.999N, 7°19938.699W), on 20 July 2020. The pH and temperature of the brine at the time of sampling were 6.7 and 41.3°C, respectively. The sample was stored cold until transported to the laboratory and immediately was sequentially filtered through 5.0- and 0.22- m m-pore polycarbonate filters (Millipore, USA), obtaining four 0.22- m mfilters that retained the desired prokaryotic fraction. The total prokaryotic DNA was obtained with phenol-chloroform-isoamyl alcohol as previously described (15, 16). Two different DNA extractions were carried out from two 0.22- m m-pore filters each. Sequence libraries were constructed from the purified prokaryotic DNA using the Novogene NGS DNA library prep set (catalog no. PT004). Metagenomic sequencing was accomplished at a read length of 2 150 bp by Novogene Europe (Cambridge, United Kingdom) on an Illumina NovaSeq 6000 platform. Totals of 72,606,726 and 49,798,159 reads were obtained for the two replicates 20IC42-1 and 20IC42-2, respectively. Read quality control was performed with the “read_qc”module as implemented in MetaWRAP v.1.3.2 using default parameters (17). The taxonomic distribution was estimated from quality filter reads using Braken v.2.8 (18). The most abundant taxa were Halorubrum (34% and 32%), Haloquadratum (10% and 13%), and Salinibacter (10% and 8%) for the 20IC42-1 and 20IC42-2 metagenomic data sets, respectively. Editor Frank J. Stewart, Montana State University Copyright © 2023 García-Roldán et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Antonio Ventosa, [email protected]. The authors declare no conflict of interest. Received 19 January 2023 Accepted 21 March 2023 Published 4 April 2023 May 2023 Volume 12 Issue 5 10.1128/mra.00039-23 1 OTHER GENETIC RESOURCES Downloaded from https://journals.asm.org/journal/mra on 07 July 2023 by 150.214.182.235. These obtained metagenomic data sets will be used to describe the phylogenetic and functional diversity of a crystallizer pond of Isla Cristina saltern and to evaluate the biochemical pathways encoded under extremely-high-salinity conditions by haloarchaea and extremely halophilic bacteria. Data availability. The sequences obtained in this project have been deposited in the NCBI Sequence Read Archive under accession no. SRR21894959 and SRR23092357 for 20IC42-1 and 20IC42-2, respectively. ACKNOWLEDGMENTS This research was supported by grant PID2020-118136GB-I00 funded by MCIN/AEI/ 10.13039/501100011033 (to A.V. and C.S.-P.). A.V. acknowledges support from the Junta de Andalucía, Spain (grants P20_01066 and BIO-213), which included FEDER funds. A.G.-R. was the recipient of a predoctoral fellowship from the Spanish Ministry of Universities. R.R.H. was the recipient of a short-stay grant “Salvador Madariaga”from the Spanish Ministry of Science, Innovation and Universities (Programa Estatal de Promoción del Talento y su Empleabilidad en I1D1I, Subprograma de Movilidad, del Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020) (PRX21/00598). B.V.-G. was the recipient of a postdoctoral grant from the Junta de Andalucía, Spain. REFERENCES 1. Ventosa A, de la Haba RR, Sánchez-Porro C, Papke RT. 2015. Microbial diversity of hypersaline environments: a metagenomic approach. Curr Opin Microbiol 25:80–87. https://doi.org/10.1016/j.mib.2015.05.002. 2. Ventosa A, Fernández AB, León MJ, Sánchez-Porro C, Rodriguez-Valera F. 2014. The Santa Pola saltern as a model for studying the microbiota of hypersaline environments. Extremophiles 18:811–824. https://doi.org/10 .1007/s00792-014-0681-6. 3. León MJ, Fernández AB, Ghai R, Sánchez-Porro C, Rodriguez-Valera F, Ventosa A. 2014. From metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium Spiribacter salinus gen. nov., sp. nov. Appl Environ Microbiol 80:3850–3857. https://doi.org/10.1128/AEM.00430-14. 4. León MJ, Vera-Gargallo B, Sánchez-Porro C, Ventosa A. 2016. Spiribacter roseus sp. nov., a moderately halophilic species of the genus Spiribacter from salterns. Int J Syst Evol Microbiol 66:4218–4224. https://doi.org/10 .1099/ijsem.0.001338. 5. León MJ, Martínez-Checa F, Ventosa A, Sánchez-Porro C. 2015. Idiomarina aquatica sp. nov., a moderately halophilic bacterium isolated from salterns. Int J Syst Evol Microbiol 65:4595–4600. https://doi.org/10.1099/ijsem.0.000619. 6. León MJ, Sánchez-Porro C, Ventosa A. 2017. Marinobacter aquaticus sp. nov., a moderately halophilic bacterium from a solar saltern. Int J Syst Evol Microbiol 67:2622–2627. https://doi.org/10.1099/ijsem.0.001984. 7. López-Hermoso C, de la Haba RR, Sánchez-Porro C, Ventosa A. 2018. Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns. Syst Appl Microbiol 41:159–166. https://doi.org/10.1016/j.syapm.2017 .12.001. 8. Durán-Viseras A, Ventosa A, Sánchez-Porro C. 2019. Halonotius aquaticus sp. nov., a new haloarchaeon isolated from a marine saltern. Int J Syst Evol Microbiol 69:1306–1312. https://doi.org/10.1099/ijsem.0.003309. 9. Durán-Viseras A, Andrei A-S, Ghai R, Sánchez-Porro C, Ventosa A. 2019. New Halonotius species provide genomics-based insights into cobalamin synthesis in haloarchaea. Front Microbiol 10:1928. https://doi.org/10.3389/ fmicb.2019.01928. 10. Durán-Viseras A, Sánchez-Porro C, Ventosa A. 2019. Halorientalis pallida sp. nov., an extremely halophilic archaeon isolated from a marine saltern. Int J Syst Evol Microbiol 69:3636–3643. https://doi.org/10.1099/ijsem.0 .003675. 11. Durán-Viseras A, Sánchez-Porro C, Ventosa A. 2020. Haloglomus irregulare gen. nov., sp. nov., a new halophilic archaeon isolated from a marine saltern. Microorganisms 8:206. https://doi.org/10.3390/microorganisms8020206. 12. Durán-Viseras A, Andrei A-S, Vera-Gargallo B, Ghai R, Sánchez-Porro C, Ventosa A. 2021. Culturomics-based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis. Environ Microbiol 23:3418–3434. https://doi .org/10.1111/1462-2920.15082. 13. Fernández AB, León MJ, Vera B, Sánchez-Porro C, Ventosa A. 2014. Metagenomic sequence of prokaryotic microbiota from an intermediate-salinity pond of a saltern in Isla Cristina, Spain. Genome Announc 2:e00045-14. https://doi .org/10.1128/genomeA.00045-14. 14. Fernández AB, Vera-Gargallo B, Sánchez-Porro C, Ghai R, Papke RT, Rodriguez- Valera F, Ventosa A. 2014. Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach. Front Microbiol 5:196. https://doi.org/10.3389/fmicb.2014 .00196. 15. Ghai R, Pasic L, Fernández AB, Martin-Cuadrado A-B, Mizuno CM, McMahon KD, Papke RT, Stepanauskas R, Rodriguez-Brito B, Rohwer F, Sánchez-Porro C, Ventosa A, Rodríguez-Valera F. 2011. New abundant microbial groups in aquatic hypersaline environments. Sci Rep 1:135. https://doi.org/10.1038/ srep00135. 16. Martín-Cuadrado AB, López-García P, Alba JC, Moreira D, Monticelli L, Strittmatter A, Gottschalk G, Rodríguez-Valera F. 2007. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS One 2:e914. https://doi.org/10.1371/journal.pone.0000914. 17. Uritskiy GV, DiRuggiero J, Taylor J. 2018. MetaWRAP—aflexible pipeline for genome-resolved metagenomic data analysis. Microbiome 6:158. https://doi .org/10.1186/s40168-018-0541-1. 18. Lu J, Breitwieser FP, Thielen P, Salzberg SL. 2017. Bracken: estimating species abundance in metagenomics data. PeerJ Comp Sci 3:e104. https://doi.org/10 .7717/peerj-cs.104. 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