ORIGINAL RESEARCH published: 10 December 2015 doi: 10.3389/fmicb.2015.01396 Metagenome-Based Metabolic Reconstruction Reveals the Ecophysiological Function of Epsilonproteobacteria in a Hydrocarbon-Contaminated Sulfidic Aquifer Andreas H. Keller1,2 , Kathleen M. Schleinitz2,RobertStarke3, Stefan Bertilsson4, Carsten Vogt1 and Sabine Kleinsteuber2* 1 Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany, 2 Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany, 3 Department of Proteomics, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany, 4 Department of Ecology and Genetics, Limnology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden The population genome of an uncultured bacterium assigned to the Campylobacterales (Epsilonproteobacteria) was reconstructed from a metagenome dataset obtained by whole-genome shotgun pyrosequencing. Genomic DNA was extracted from a sulfate-reducing, m-xylene-mineralizing enrichment culture isolated from groundwater of a benzene-contaminated sulfidic aquifer. The identical epsilonproteobacterial phylotype has previously been detected in toluene- or benzene-mineralizing, sulfate- reducing consortia enriched from the same site. Previous stable isotope probing (SIP) 13 Edited by: experiments with C6-labeled benzene suggested that this phylotype assimilates Marc Strous, benzene-derived carbon in a syntrophic benzene-mineralizing consortium that uses University of Calgary, Canada sulfate as terminal electron acceptor. However, the type of energy metabolism and the Reviewed by: Lisa Gieg, ecophysiological function of this epsilonproteobacterium within aromatic hydrocarbon- University of Calgary, Canada degrading consortia and in the sulfidic aquifer are poorly understood. Annotation of the Trinity L. Hamilton, epsilonproteobacterial population genome suggests that the bacterium plays a key role University of Cincinnati, USA *Correspondence: in sulfur cycling as indicated by the presence of an sqr gene encoding a sulfide quinone Sabine Kleinsteuber oxidoreductase and psr genes encoding a polysulfide reductase. It may gain energy [email protected] by using sulfide or hydrogen/formate as electron donors. Polysulfide, fumarate, as well Specialty section: as oxygen are potential electron acceptors. Auto- or mixotrophic carbon metabolism This article was submitted to seems plausible since a complete reductive citric acid cycle was detected. Thus the Microbial Physiology and Metabolism, bacterium can thrive in pristine groundwater as well as in hydrocarbon-contaminated a section of the journal Frontiers in Microbiology aquifers. In hydrocarbon-contaminated sulfidic habitats, the epsilonproteobacterium Received: 01 October 2015 may generate energy by coupling the oxidation of hydrogen or formate and highly Accepted: 23 November 2015 abundant sulfide with the reduction of fumarate and/or polysulfide, accompanied by Published: 10 December 2015 efficient assimilation of acetate produced during fermentation or incomplete oxidation Citation: Keller AH, Schleinitz KM, Starke R, of hydrocarbons. The highly efficient assimilation of acetate was recently demonstrated 13 Bertilsson S, Vogt C by a pulsed C2-acetate protein SIP experiment. The capability of nitrogen fixation as and Kleinsteuber S (2015) indicated by the presence of nif genes may provide a selective advantage in nitrogen- Metagenome-Based Metabolic Reconstruction Reveals depleted habitats. Based on this metabolic reconstruction, we propose acetate capture the Ecophysiological Function and sulfur cycling as key functions of Epsilonproteobacteria within the intermediary of Epsilonproteobacteria in a Hydrocarbon-Contaminated Sulfidic ecosystem metabolism of hydrocarbon-rich sulfidic sediments. Aquifer. Front. Microbiol. 6:1396. Keywords: Campylobacterales, sulfur cycling, rTCA cycle, acetate assimilation, anaerobic hydrocarbon doi: 10.3389/fmicb.2015.01396 degradation, nitrogen fixation, niche adaptation, intermediary ecosystem metabolism Frontiers in Microbiology | www.frontiersin.org 1 December 2015 | Volume 6 | Article 1396 Keller et al. Epsilonproteobacteria in a Sulfidic Aquifer INTRODUCTION under sulfate-reducing conditions in a syntrophic, benzene- mineralizing consortium and was shown to be distantly related Representatives of the Epsilonproteobacteria inhabit a broad to the genus Sulfurovum (Kleinsteuber et al., 2008; Herrmann spectrum of environments like mammalian digestive systems et al., 2010). An identical phylotype (in the following named (Wolin et al., 1961; Engberg et al., 2000), brackish water (Brettar as Zeitz epsilonproteobacterium) was consistently detected et al., 2006), hydrothermal sediments (Inagaki et al., 2003), in various toluene-degrading (Müller et al., 2009; Jehmlich or subsurface systems (Watanabe et al., 2000; Gittel et al., et al., 2010; Kuppardt et al., 2014)andm-xylene-degrading 2012; Hubert et al., 2012; Handley et al., 2013). Previously (Bozinovski et al., 2012) sulfate-reducing enrichment cultures obtained isolates have been described as chemolithoautotrophs from the same site and remained abundant after prolonged (Takai et al., 2006; Sievert et al., 2008) fixing carbon via the incubation. The closest relatives of this phylotype based on 16S reductive tricarboxylic acid (rTCA) cycle (Hügler et al., 2005). rRNA gene sequences were found in pristine sulfidic springs and Notably, they are recognized as key players in sulfidic habitats caves (Porter and Engel, 2008; Jones et al., 2010). A DNA-SIP 13 (Campbell et al., 2006) capable of oxidizing sulfide, sulfur or experiment with C6-benzene revealed significant labeling of thiosulfate, or using elemental sulfur/polysulfide as terminal the Zeitz epsilonproteobacterium, besides the putative initial electron acceptors. Furthermore, oxygen, nitrate, and fumarate benzene degrader, a clostridial phylotype assigned to the genus can be electron acceptors (Campbell et al., 2006; Miller et al., Pelotomaculum (Herrmann et al., 2010). 2007). Besides reduced inorganic sulfur compounds, hydrogen, However, the respective protein-SIP experiment did not or organic substances such as malate and formate were shown to confirm labeling of the Zeitz epsilonproteobacterium whereas be electron donors (Campbell et al., 2006). The broad spectrum of benzene assimilation by the initial degrader Pelotomaculum habitats where Epsilonproteobacteria can be found is underlined sp. was verified (Taubert et al., 2012). Likewise, protein-SIP 13 by their capability to grow under aerobic, microaerobic, or with methyl-labeled m-xylene (1,3-dimethyl- C2-benzene) did anoxic conditions. A model organism representing the metabolic not lead to a labeling of epsilonproteobacterial peptides within versatility of this proteobacterial class is Wolinella succinogenes.It the m-xylene-degrading enrichment culture (Bozinovski et al., couples anaerobic fumarate or nitrate respiration with hydrogen, 2012, 2014). Thus, the Zeitz epsilonproteobacterium seems sulfide, or formate oxidation (Kröger et al., 2002; Kern and to be not primarily involved in hydrocarbon degradation, Simon, 2009) but can also grow under limited oxic conditions despite being consistently present in the respective consortia (Wolin et al., 1961). During the last decade, research focused in varying relative abundances. We hypothesize that it uses on Epsilonproteobacteria thriving in marine systems such as metabolites from hydrocarbon degradation under sulfate- hydrothermal vents (Miroshnichenko et al., 2004; Nakagawa reducing conditions, but the type of energy metabolism and its et al., 2007; Schauer et al., 2011; Stokke et al., 2015)orpelagic specific ecophysiological role in the consortia have remained oxic-anoxic interfaces (Grote et al., 2012) as well as in terrestrial unknown so far. To shed light on the ecological niche and sulfidic caves and springs (Engel et al., 2004; Porter and Engel, metabolic function of Epsilonproteobacteria in hydrocarbon-rich 2008; Jones et al., 2010; Rossmassler et al., 2012; Hamilton et al., sulfidic environments, we aimed at a metabolic reconstruction 2015), or mud volcanos (Green-Saxena et al., 2012)wherethey of the Zeitz epsilonproteobacterium based on genome-centric are thought to be mainly involved in the oxidation or reduction metagenomics. of sulfur compounds. Recently, Epsilonproteobacteria were also found to be abundant in anoxic hydrocarbon-rich habitats like oil reservoirs MATERIALS AND METHODS (Hubert et al., 2012), phenol-degrading methanogenic sludge (Zhangh et al., 2005), petroleum-contaminated soil (Kasai et al., DNA Isolation and Whole Genome 2005), and hydrocarbon-degrading sulfate-reducing enrichment Amplification cultures (Kleinsteuber et al., 2008; Müller et al., 2009; Jehmlich Cells originated from a batch culture used as a control in a growth et al., 2010; Pilloni et al., 2011; Bozinovski et al., 2012; Kuppardt experiment with an m-xylene-degrading, sulfate-reducing batch et al., 2014). The metabolism of Epsilonproteobacteria in anoxic culture (Herrmann et al., 2009; Bozinovski et al., 2012, 2014). hydrocarbon-contaminated subsurface systems and especially The medium in the control culture did not contain any in sulfate-reducing consortia is poorly understood as they are organic carbon source. During the experiment, phylogenetic neither known to perform dissimilatory sulfate reduction nor to composition was determined by terminal restriction fragment degrade aromatic or aliphatic