Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone Patricia L. Tavormina, 1 * William Ussler III, 2 Joshua A. Steele, 1 Stephanie A. Connon, 1 Martin G. Klotz 3 and Victoria J. Orphan 1 * 1 Division of Geological and Planetary Sciences, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA. 2 Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA. 3 Department of Biology, University of North Carolina, Charlotte, NC 28223, USA. Summary Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine envi- ronment, suggesting widespread potential for oxida- tion of reduced substrates. Here, we used the well- defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differ- ences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating signifi- cantly with reduced environmental oxygen concentra- tions and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene- utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO- encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affili- ated phylogenetically with the recently described Cu-MMO ‘pXMO’, and Group Z clustered with Cu- MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced mol- ecules and advance our understanding of the micro- organisms involved in methane cycling in the OMZ environment. Introduction The marine methane cycle impacts diverse processes ranging from the establishment of chemosynthetic com- munities at seafloor methane seeps to the flux of methane between the ocean and the atmosphere (for reviews see Levin, 2005; Reeburgh, 2007). In sufficiently oxic ocean waters, the enzyme complex particulate methane monooxygenase (pmoCAB, pMMO), a member of the copper-containing membrane-bound monooxygenase (Cu-MMO) superfamily, catalyses the aerobic oxidation of methane. Along continental margins, two pMMO- encoding phylogenetic groups termed OPU1 and OPU3 (Hayashi et al., 2007), also known as deep-sea 5 and deep-sea 3 clusters (Luke and Frenzel, 2011), are com- monly recovered in molecular gene surveys targeting aerobic methanotrophs in the ocean (Jensen et al., 2008; Wasmund et al., 2009; Tavormina et al., 2010; Kessler et al., 2011), and expression of pmoCAB for group OPU3 has recently been demonstrated in a metatranscriptome from the Guaymas Basin (Lesniewski et al., 2012). Taxo- nomically, groups OPU1 and OPU3 affiliate with Methylo- coccales phylotypes, and they may comprise a broadly distributed pelagic sink for methane along continental margins at water depths below ~100 m (Tavormina et al., 2010). Although several studies have begun to examine the global distributions of phylogenetic groups OPU1 and OPU3, the extent to which these groups vary in relation to geochemical parameters including spatial methane and oxygen concentrations has not been described in detail. Such variance may reflect the methane-oxidizing potential of particular zones in the water column, and ascertaining this variance could reveal distinct ecological strategies employed by these groups. Molecular gene surveys of the water column have also identified substantially sequence-divergent marine Cu-MMO-encoding sequences (Redmond et al., 2010; Tavormina et al., 2010), and the genomes of select Received 6 September, 2012; accepted 3 December, 2012. *For correspondence. E-mail vorphan@gps.caltech.edu; pattytav@ gps.caltech.edu; Tel. (+1) 626 395 1786; Fax (+1) 626 683 0621. Environmental Microbiology Reports (2013) doi:10.1111/1758-2229.12025 © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd