The ISME Journal (2015) 9, 16–31 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists Marı´a Gomariz1,2, Manuel Martı´nez-Garcı´a2, Fernando Santos2, Francisco Rodriguez3, Salvador Capella-Gutie´rrez4, Toni Gabaldo´n4,5, Ramon Rossello´-Mo´ra6, Inmaculada Meseguer1 and Josefa Anto´n2 1Department of Materials, Optics and Electronics, University Miguel Herna´ndez of Elche, Alicante, Spain; 2Department of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, Spain; 3Departament of Applied Mathematics, University of Alicante, Alicante, Spain; 4Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain; 5Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain and 6Marine Microbiology Group, Department of Ecology and Marine Resources, Institut Mediterrani d’Estudis Avanc¸ats IMEDEA (CSIC-UIB), Esporles, Spain The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to 440%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group. The ISME Journal (2015) 9, 16–31; doi:10.1038/ismej.2014.95; published online 13 June 2014 Introduction and sodium chloride occurs. This salinity gradient is accompanied by a succession of different groups Multi-pond solar salterns consist of a series of of microorganisms, from marine to extremely interconnected ponds with increasing salinity, halophilic microbiota, each adapted to a salinity from seawater level to sodium chloride saturation range. (Benlloch et al., 2002; Pasˇic´ et al., 2005; Oh et al., Saltern ponds have been traditionally considered 2010; Boujelben et al., 2012). In continuously ‘steady-state’ systems because of the stability of both operated salterns, such as the Santa Pola (Spain) salinity and microbial composition of the different facilities studied here, seawater is pumped ponds (Guixa-Boixereu et al., 1996; Ghai et al., through a set of shallow ponds where sequential precipitation of calcium carbonate, calcium sulfate 2011). Although it is generally assumed that the composition of saltern microbial communities is ‘fairly stable over time even when subjected to Correspondence: J Anto´n, Department of Physiology, Genetics, environmental perturbations’ (Gasol et al., 2004; and Microbiology, University of Alicante, Alicante 03080, Spain. Ghai et al., 2011), most studies do not include E-mail: [email protected] Received 7 October 2013; revised 8 May 2014; accepted 10 May different time points and provide only snapshots of 2014; published online 13 June 2014 the microbial diversity without addressing in situ Community dynamics in hypersaline environments M Gomariz et al 17 temporal dynamics. However, some studies hint at a further characterized this bacterial group by means more dynamic picture of these systems. A metage- of database search and single-cell genome analyses. nomic analysis of the San Diego salterns community (Rodriguez-Brito et al., 2010) using samples har- vested at various time points separated by 1 day to Materials and methods 41 year showed that most abundant microbial Sample collection ‘species’ persisted, but their relative abundances Five contiguous ponds of Bras del Port salterns and microdiversity changed over time. Notwith- (Santa Pola, Spain, 381 120 N, 01 360 W) were standing, this study only included two time points analyzed in this study (Figure 1): two medium (01 July 2004 and 28 November 2005) for ponds of concentrators (CM1 and CM2), a brine concentrator medium high salinities (above 18%), and thus its (CCAB), and two crystallizers (CR30 and CR41). conclusions cannot be extrapolated to wider tem- Samples were collected on the following dates in poral ranges for this kind of environments. More 2006: 23 January (JN), 7 March (MR), 26 April (AP), recently, a study of the Tunisian solar salterns of 6 June (J1), 27 June (J2), 25 July (JL), 5 September Sfax (Boujelben et al., 2012) also found a temporal (SP), 2 October (OC) and 28 November (NV). succession of phylotypes in medium salinity (around 20%) ponds, with some specific bacterial sequences appearing only at some time points along Environmental parameters the year. Temperature, pH and salinity were measured in situ In order to gain insight into the changes experi- while solar radiation and rainfall data were pro- enced by microbial communities in saltern ponds vided by the meteorological station of Bras del we have undertaken a comprehensive seasonal Port. Chemical analyses (sulfate, chloride, study of five ponds of medium and high salinity carbonate, phosphate, sodium, potassium, calcium, (from 18% to 40% total salts) from Santa Pola solar magnesium and ammonium) were carried out by salterns. The prokaryotic community of these saltern atomic absorption spectrometry (Unicam SOLAAR ponds has been studied for decades using several 969, Unicam Ltd., Cambridge, UK) at the Depart- approaches, depending on the available tools, from ment of Agrochemistry and Environment (Univer- classical cultivation to culture-independent techni- sity Miguel Herna´ndez de Elche, Spain). ques. The lower-medium (15–25%) salinity ponds are inhabited by members of the Proteobacteria, Cyanobacteria, Bacteroidetes or haloarchaea-like CARD-FISH Halosarcina or Halorubrum, whereas crystallizers Archaeal and bacterial counts were measured (ponds where NaCl precipitates) are dominated by following an optimized CARD-FISH protocol mod- high numbers of the archaeon Haloquadratum ified from Pernthaler et al. (2004). Samples were walsbyi and extremely halophilic Bacteroidetes, fixed with formaldehyde (7% final concentration; such as Salinibacter ruber, that account for up to Sigma, St Louis, MO, USA) at 4 1C for 16 h, diluted 75% and 18% of the total community, respectively (Anto´n et al., 1999; Benlloch et al. 2002; Burns et al. 2004; Estrada et al., 2004; Ghai et al., 2011; Narasingarao et al., 2012; Zhaxybayeva et al., 2013). Recently, the use of Next-Generation Sequen- cing (NGS) metagenomics and single-cell genomics has led to the discovery of new abundant groups (Ghai et al., 2011; Narasingarao et al., 2012), such as a group of low GC Actinobacteria in low and intermediate salinities, and a new archaeal linage of Nanohaloarchaea in crystallizers. Our results indicate that saltern microbiota is dynamic and within a given pond microbial com- position can change considerably along the year. In addition, total salinity is not the only factor structuring microbial assemblages in hypersaline environments as ponds of the same salinity har- bored different microbial communities. Some micro- bial phylotypes were strongly associated with specific environmental conditions while others Figure 1 Aerial photograph of Bras del Port saltern ponds, Santa were very widely distributed both spatially and Pola, Alicante. Water is pumped from the sea to preconcentrators, temporally. Especially noteworthy was the case of medium concentrators, a brine concentrator and finally to the crystallizers. The ponds analyzed in this study are framed with an uncultured Bacteroidetes phylotype that was dotted white lines: CM1 and CM2 (medium concentrators), CCAB present along the whole salinity gradient and could (brine concentrator) and CR30 and CR41 (crystallizers). Arrows thus constitute a generalist strategist. Here, we have indicate the water circuit. The ISME Journal Community dynamics in hypersaline environments M Gomariz et al 18 (1/1000, 1/500 and 1/200) in 1X phosphate-buffered water. The extracted nucleic acids were analyzed by saline (8 g NaCl, 1.44 g Na2HPO4, 2 g KCl, 2 g electrophoresis in 0.8% agarose gels (LE, FMC KH2PO4, pH 7.0) and filtered through 0.2-mm pore Bioproducts, Rockland,