The ISME Journal (2014) 8, 1510–1521 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap) Costantino Vetriani1,2,4, James W Voordeckers1,2,4,5, Melitza Crespo-Medina1,2,6, Charles E O’Brien1,2, Donato Giovannelli1,2,3 and Richard A Lutz2 1Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA; 2Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA and 3Institute of Marine Science - ISMAR, National Research Council of Italy, CNR, Ancona, Italy Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydro- thermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body. The ISME Journal (2014) 8, 1510–1521; doi:10.1038/ismej.2013.246; published online 16 January 2014 Subject Category: Microbial ecology and functional diversity of natural habitats Keywords: Epsilonproteobacteria; nitrate reduction; deep-sea vents; napA Introduction or ammonium (Blochl et al., 1997; Vetriani et al., 2004; Voordeckers et al., 2008; Perez-Rodriguez The contribution of deep-sea vent microorganisms et al., 2010, 2012). In most cases, nitrate reduction to the sulfur cycle has been extensively investigated is coupled to the oxidation of molecular hydrogen (Jannasch, 1995; Karl, 1995; Reysenbach et al., (and less frequently of formate or thiosulfate), and 2002). However, the isolation and characterization these organisms have a shorter doubling time when of a number of novel bacteria and archaea from nitrate, rather than sulfur, is supplemented as the deep-sea hydrothermal vents revealed that most of terminal electron acceptor (Vetriani et al., 2004; them can use nitrate in addition to sulfur as their Voordeckers et al., 2005; Perez-Rodriguez et al., terminal electron acceptor. During growth, these 2010, 2012). In particular, most of the Epsilonpro- microorganisms reduce nitrate to dinitrogen (Takai teobacteria isolated from deep-sea hydrothermal et al., 2004; Nakagawa et al., 2005; Takai et al., 2006) vents conserve energy by reducing nitrate (reviewed in Campbell et al., 2006). Epsilonproteobacteria Correspondence: C Vetriani, Institute of Marine and Coastal have been found to be abundant in geographically Sciences, Rutgers University, 71 Dudley Road, New Brunswick, distant deep-sea vent sites, and colonization experi- NJ 08901, USA. ments that were carried out in the vicinity of active E-mail: [email protected] 4These authors contributed equally to this work. deep-sea vents revealed that between 66 and 98% of 5Current address: Department of Botany and Microbiology, the microorganisms associated with these substrates Institute for Environmental Genomics, University of Oklahoma, belonged to this group of bacteria (Lopez-Garcia Norman, OK 73019, USA et al., 2003; Takai et al., 2003; Alain et al., 2004). 6Current address: Department of Biology, East Carolina University, Greenville, NC 27858, USA Overall, these observations indicate that Epsilon- Received 14 September 2013; revised 1 December 2013; accepted proteobacteria represent a large fraction of the 3 December 2013; published online 16 January 2014 primary producers at deep-sea hydrothermal vents Nitrate reductase in vent Epsilonproteobacteria C Vetriani et al 1511 at temperatures between 25 and 60 1C, and imply several Epsilonproteobacteria isolated from marine that the use of nitrate as a terminal electron acceptor geothermal environments (Inagaki et al., 2003; in these environments may be more common than Voordeckers et al., 2005; Nakagawa et al., 2007; previously thought. Campbell et al., 2009; Sikorski et al., 2010; Data on the nitrogen chemistry of hydrothermal Giovannelli et al., 2011). The genome sequence of fluids indicate a depletion of both nitrate and nitrite these organisms showed that Epsilonproteobacteria in vent fluids. Nitrate, however, is present in bottom encode the periplasmic nitrate reductase, NapA, and seawater (Millero, 1996) and it is therefore available not for the membrane-bound enzyme, NarG (in to be used by vent bacteria both as an electron contrast to, for example, E. coli, which encodes acceptor and as a nitrogen source. More recently, both enzymes). Bourbonnais et al. (2012a, b) found that the rates for The napA gene represents the core entity of the nitrate reduction to dinitrogen gas and ammonium periplasmic nitrate reductase gene cluster. The in fluids from diffuse flow vents on the Juan de Fuca napDAB genes encode the molybdopterin-contain- Ridge were up to B1000 and 150 nmol N l À 1 per day, ing reductase (NapA), the diheme component of respectively, and that the isotopic composition of the periplasmic electron transfer system (NapB, the nitrate and ammonium suggested a role for vent redox partner of NapA) and a putative chaperone microorganisms in nitrogen cycling. involved in the maturation of NapA (NapD). Prokaryotes are able to synthesize three distinct In Epsilonproteobacteria, the role of individual types of nitrate reductases. All three enzymes products of the nap gene cluster has been investigated reduce nitrate to nitrite, but they are involved in in W. succinogenes (Kern et al., 2007). Consistent different physiological processes (reviewed in Lin with the lack of the NapC (Simon et al., 2003), the and Stewart, 1998; Moreno-Vivian et al., 1999; electron transfer to NapAB in W. succinogenes is Potter et al., 2001; Richardson et al., 2001). The mediated by the NapGH membrane-bound iron– Nas enzyme is located in the cytoplasm and sulfur periplasmic quinone dehydrogenase complex participates in nitrogen assimilation, whereas the (Kern and Simon, 2008). NapL is an iron–sulfur other two nitrate reductases (Nar and Nap) are both periplasmic protein present in all epsilonproteobac- linked to respiratory electron transport systems. The terial genomes. Although the function of NapL and Nar enzyme is a three-subunit complex attached to NapF is currently unclear, recent data indicate that the cytoplasmic side of the membrane. The Nap in W. succinogenes the NapGHF complex is enzyme is a two-subunit complex located in the involved in menaquinol oxidation, electron transfer periplasmic space of Gram-negative bacteria. All to periplasmic NapA and maturation of the cyto- three types of nitrate reductases contain molybde- plasmic NapA precursor (Kern et al., 2007; Kern and num bound to the bis-molybdopterin guanine Simon, 2009b). dinucleotide cofactor at the active site. However, Although surveys of genes involved in nitrate although the assimilatory enzyme (Nas) uses reduction have been carried out in soils and NAD(P)H or ferredoxin as reductant, the respiratory estuarine environments (Bru et al., 2007; Smith Nar and Nap enzymes ultimately take electrons from et al., 2007; Dong et al., 2009), the distribution and the membranous quinone pool. diversity of the nitrate reductase in natural micro- The periplasmic nitrate reductase (NapA, bial communities from deep-sea geothermal envir- encoded by the napA gene) has been described in onments are scarce. Recently, Perez-Rodriguez et al. several different organisms, and its physiological (2013) identified Gamma- and Alphaproteobacteria- functions may vary in different bacteria (Moreno- related, membrane-bound nitrate reductases, NarG, Vivian et al., 1999; Potter et al., 2001; Richardson in vent microbial communities. However, there is no et al., 2001). Although in some bacteria NapA has a information on the distribution and diversity of the dissimilatory function to maintain redox balance periplasmic nitrate reductase, NapA, in deep-sea during growth on highly reduced carbon sources environments. In this study we have reconstructed (for example, in Rhodobacter and Paracoccus spp.; the structure of the nap gene cluster in Epsilonpro- Richardson et al., 2001), in some enteric and rumen teobacteria isolated from deep-sea hydrothermal bacteria, such as Escherichia coli and Wolinella vents whose genomes have been sequenced; we succinogenes, NapA is expressed during