Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment María-Eugenia Guazzaroni, Verónica Morgante, Salvador Mirete and José E. González-Pastor* Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid, Spain. Summary Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizo- sphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the tran- scriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowl- edge about the different strategies used by microor- ganisms to survive under extremely acid conditions. Introduction Acidic environments both natural and artificial occur in nature, including sulfidic mine areas and marine volcanic vents (Baross and Hoffman, 1985; Johnson and Hallberg, 2005). Microorganisms that thrive in those habitats are termed acidophiles and are able to grow at a pH of less than 3. The genomes of these organisms are endowed with a diverse repertoire of genes encoding for different mechanisms of adaptation at low pH, and the access to the gene pool of acidophiles would allow the identification and isolation of novel genetic determinants with a poten- tial use in biotechnology. However, the exceptional life- style presented by these organisms also makes difficult to use classical growth-dependent approaches as most of them cannot be cultivated (Rappe and Giovannoni, 2003; Baker-Austin and Dopson, 2007). Fortunately, the advent of metagenomics has provided a key tool that allows accessing the genetic wealth of the genomes from uncultured microorganisms (Streit and Schmitz, 2004; Fernandez-Arrojo et al., 2010). Several processes in industry require enzymes or biocatalysts that can work in mildly acid conditions such as the bioconversion of ligno- cellulosic material to bioethanol (Wyman et al., 2005) and biohydrogen production by dark fermentation (Chong et al., 2009). Therefore, obtaining a microorganism able to carry out those procedures efficiently at low pH is an imperative goal in biotechnology. When bacteria are exposed to acidic conditions, protons may enter into the cell, strongly reducing the cytoplasmic pH (Richard and Foster, 2004). The detrimen- tal effects of acidic pH, including protein unfolding and uncoupling of oxidative phosphorylation (Goto et al., 1990; Richard and Foster, 2004; Hong et al., 2005), lead to disruption of regular biological processes and damage to cellular structures, eventually causing cell death (Small et al., 1994; Smith, 2003). Microorganisms have devel- oped a diversity of mechanisms to survive to extremely low pH. For instance, several bacteria such as Escherichia coli express amino acid decarboxylase systems in which a reductive decarboxylation of the sub- strate (usually glutamate, arginine or lysine) consumes a proton and thus the concentration of free protons decreases in the cytoplasm (Foster, 2004). This process is followed by the extrusion of the product from the cyto- plasm by a dedicated antiporter that also imports the original amino acid. Additionally, some bacteria are able to directly export protons as a consequence of ATP hydroly- sis using the proton-translocating F 1F0 ATPase (Tucker et al., 2002) or can reverse their cytoplasmic membrane Received 1 August, 2012; revised 3 October, 2012; accepted 6 October, 2012. *For correspondence. E-mail gonzalezpje@cab.inta- csic.es; Tel. (+34) 91 5206434; Fax (+34) 91 5201074. Environmental Microbiology (2013) 15(4), 1088–1102 doi:10.1111/1462-2920.12021 © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd