The ISME Journal (2016) 10, 299–309 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea Nicolás Rascovan1, Javier Maldonado2, Martín P Vazquez1 and María Eugenia Farías2 1Instituto de Agrobiotecnología de Rosario (INDEAR), Ocampo 210 bis (2000), Predio CCT Rosario, Santa Fe, Argentina and 2Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto de Procesos Industriales Microbiológicos (PROIMI), CCT, CONICET, San Miguel de Tucumán, Tucumán, Argentina Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics. The ISME Journal (2016) 10, 299–309; doi:10.1038/ismej.2015.109; published online 3 July 2015 Introduction evolutionary analyses of key enzymes involved in these processes (Schoepp-Cothenet et al., 2012). Life forms use various bioenergetic strategies to Such is the case of arsenite (As(III)), which is obtain free energy from the environment and trans- proposed as a candidate electron donor, feeding form it into electrochemical gradients that generate bioenergetic chains in primordial life based on ATP. The first microorganisms likely used inorganic phylogenetic analysis of large subunit of arsenite compounds to fuel bioenergetic redox reactions. The oxidases (AioA) and paleogeochemical analyses most-accepted hypothesis is that energy obtained (Lebrun et al., 2003; van Lis et al., 2013; Sforna from the oxidation of H2 and the reduction of CO2 et al., 2014). In fact, the arsenic seems to have been were the processes used by the common ancestor of present at much higher concentrations in the ancient Bacteria and Archaea known as last universal Earth’s crust than it is today (Oremland et al., 2009). common ancestor (Martin et al., 2008). However, The characteristics and biochemical nature of the several other inorganic substrates seem to have ancient arsenic metabolism is subject of an ongoing been used in bioenergetic redox reactions in the debate (Kulp et al., 2008; Schoepp-Cothenet et al., last universal common ancestor based on the 2009). Members of both Bacteria and Archaea encode aio genes for As(III) oxidation, which could be used for chemolithotrophic growth (van Lis et al., Correspondence: ME Farías, Laboratorio de Investigaciones Micro- biológicas de Lagunas Andinas (LIMLA), PROIMI-CONICET, 2013). Some Bacteria have also genes for dissim- Avenida Belgrano y Pje. Caseros, Tucumán 4000, Argentina. ilatory arsenate (As(V)) reduction (arr genes), asso- E- mail: [email protected] ciated to anaerobic respiration using arsenate as or N Rascovan, Instituto de Agrobiotecnología de Rosario (INDEAR), terminal electron acceptor (Duval et al., 2008). It has Ocampo 210 bis, 2000, Predio CCT Rosario, Santa Fe, Argentina. E-mail: [email protected] been proposed that anaerobic arsenate respiration is Received 15 April 2015; revised 14 May 2015; accepted a more recent metabolism than arsenite oxidation, 20 May 2015; published online 3 July 2015 emerged once oxygen became abundant at the Arsenic bioenergetics in haloarchaea biofilms N Rascovan et al 300 atmosphere (van Lis et al., 2013). Although Archaea used for sequencing was extracted from a mixture of and Bacteria are able to grow anaerobically on three triplicate using PowerBiofilm DNA Isolation arsenate, the arrA gene (the only well-characterized Kit (MO BIO Laboratories, Inc.) from 0.5 g of enzyme that catalyze respiratory arsenate reduction) collected biofilm and processed according to manu- has only been found in Bacteria. facturer’s indications. Haloarchaea are the most studied Archaea (phy- lum: Euryarchaeota) and a very well studied group of microorganisms. They are free-living halophiles, Scanning electron microscopy aerobic and sometimes facultative anaerobic, Samples for electron microscopy analyses were heterotrophs that in some cases synthetize ATP prepared scraping biofilm-associated crystals and yielding energy from light using bacteriorhodopsins fixed overnight at 4 °C in a Karnovsky fixative (Falb et al., 2008). Oxygen is poorly soluble at made with formaldehyde (8% v/v), glutar-aldehyde high salt concentrations and anaerobic growth of (16% v/v) and phosphate buffer (pH 7). Fixed haloarchaea has been observed using nitrate, samples were washed three times with phosphate dimethylsulfoxide, trimethylamine N-oxide and buffer and CaCl2 for 10 min, and fixed with osmium fumarate as electron acceptors, some of which are tetroxide (2% v/v) overnight. Afterward, samples probably remnants of the ancestral haloarchaea were washed twice with ethanol (30% v/v) for 10 metabolisms that prevailed when Earth’s atmosphere min, dried at a critical point, and sputtered with gold. was still anoxic (Oren, 2013). Here we report the Specimens were observed under vacuum using a Zeiss discovery of haloarchaea forming biofilms in nature Supra 55VP (Carl Zeiss NTS GmbH, Oberkochen, in a high altitude soda lake under high arsenic Germany) at the Centro Integral de Microscopía concentrations, which are most likely using arsenic Electrónica (CIME, San Miguel de Tucumán, as a bioenergetic substrate to sustain growth. Argentina). Materials and Methods Isolation of Halorubrum strains from diamante lake The medium used for the isolation of Halorubrum Sample processing sp. AD156 and Halorubrum sp. BC156 was WSJ Biofilm samples were collected from Laguna Dia- broth, a medium based on that used to isolate mante, located inside the Galan Volcano crater and Salinibacter ruber (Anton et al., 2002; pH 8.5), − 1 − 1 placed at 4589 m above sea level (coordinates: 26˚00’ containing: NaCl 252.16 g l ; MgCl2.6H2O 0.5 g l ; ˚ ’ − 1 − 1 51.04"S , 67 01 46.42"O Figure 1). Galan Volcano is MgSO4·7H2O 0.5 g l ; KNO3 1.011 g l ; KCl 5.84 placed at Catamarca province in Argentina and reach gl− 1; Peptone (Oxoid) 5 g l − 1; Yeast extract (Oxoid) 5912 m above sea level. It is one of the super- 1gl− 1; Trace element solution 1 ml (preparation: volcanoes of the World, which hosted several large- HCl, 25% 7.7 M 10 ml, FeCl2 1.5 g, ZnCl2 70 mg, volume explosive eruptions from upper Miocene to MnCl2·4H2O 100 mg, H3BO3 6 mg, CoCl2·2H2O Pleistocene (between 7 and 4 Ma ago), (Ruggieri 190 mg, CuCl2·2H2O 2 mg, NiCl2·6H2O 24 mg, et al., 2011; Soler, 2007). Galan Volcano has the Na2MoO4·2H2O 36 mg), to a final volume of 1 l with largest uncovered caldera in the World with 40-km distilled water. Solid media were prepared with diameter. Owing to the high altitude and volcanic 1.45% agar (Oxoid). Isolation was done from 5 g of influence, Diamante Lake presents multiple extreme red biofilm sampled from Diamante Lake, which was conditions that include: high UV radiation, salinity inoculated in fresh WSJ broth and left for enrichment and arsenic, low O2 pressure and a hydrothermal during 5 days (37 ºC; 160 r.p.m.). After incubation, vent input. Three independent samples were taken 100 μl of enriched media was plated on solid WSJ from Diamante Lake Red Biofilms and water that media and incubated aerobically at 37 °C for 12 days. were collected in sterile plastic bags and flasks in Pure colonies were finally retrieved and PCR of the February 2012. Samples for scanning electron micro- 16 S rRNA gene using the 344 F (5′-ACGGGGYGCAG scopy (SEM) and for water chemical analyses were CAGGCGCGA-3′) and 915 R (5′-GTGCTCCCCCGCCA stored in the dark at 4 °C and processed within 1– ATTCCT-3′) primers was performed to identify the 2 weeks. Conductivity, temperature, pH, dissolved taxonomy of isolates. O2 and total dissolved solids were measured in situ with a multiparameter probe HANNA HI 9828. Nutrient, ions and general chemical analyses were High-throughput DNA Sequencing performed in Estación Experimental Obispo Colom- The total DNA sample obtained from DLRB was used bres, Tucumán, Argentina (http://www.eeaoc.org.ar/) to prepare a Metagenomic Shotgun Library. A Rapid a chemical IRAM-certified laboratory. Samples for library was constructed from 500 ng of DNA follow- DNA extraction were frozen in liquid nitrogen, ing Roche 454-FLX manual’s instructions. stored in the dark and processed within a week. The V4 hypervariable region of the 16 S rDNA Water samples were delivered to the facility Estación gene was amplified using F563-R802 universal Experimental Obispo Colombres, Tucumán, Argen- primers that are reliable amplifying Bacteria but tina for chemical analyses. Total metagenomic DNA not Archaea. Primer sequences were obtained from The ISME Journal Arsenic bioenergetics in haloarchaea biofilms N Rascovan et al 301 a c b Figure 1 General description of Diamante Lake Red Biofilms (DLRB). (a) Photography of DLRB obtained from the bottom of the lake.