Non-cyanobacterial nifH phylotypes in the North Pacific Subtropical Gyre detected by flow-cytometry cell sorting Deniz Bombar,* Kendra A. Turk-Kubo, Julie Robidart, Brandon J. Carter and Jonathan P. Zehr Ocean Sciences Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA. Summary In contrast to cyanobacteria, the significance of bac- teria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur as contamination from reagents and other sources, com- plicating the detection and interpretation of environ- mental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell popula- tions sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photohetero- trophic non-cyanobacterial nifH phylotypes previ- ously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations, and was detected in water column samples using quantitative PCR (qPCR), with 60% of samples detected in the > 10 mm size fraction in addition to the 0.2–10 mm size fraction. A novel cluster 3-like nifH sequence was also recovered from discrete cell sorts and detected by qPCR in environ- mental samples. This approach enables the detection of rare nifH phylotypes, identifies possible associa- tions with larger cells or particles and offers a possi- ble solution for distinguishing reagent contaminants from real microbial community components. Introduction Dinitrogen (N 2) fixation is a key component of the marine nitrogen cycle, representing an important external source of biologically available nitrogen to the ocean (Gruber and Galloway, 2008). Research on the identity, diversity and autecology of oceanic N2-fixing prokaryotes (diazotrophs) has mainly focused on cyanobacterial species so far (Capone et al., 1997; Carpenter et al., 1999; Karl et al., 2002; Zehr, 2011). A pivotal tool in these discoveries has been the analysis of the diversity and distribution of the nifH gene, which encodes the iron-containing subunit of the nitrogenase enzyme (Zehr and McReynolds, 1989; Zehr et al., 2001). Since the late 1980s, nifH has become the most widely used marker gene for analysing diazo- troph diversity and ecology in aquatic and terrestrial eco- systems, with several tens of thousands of partial nifH sequences currently in public sequence databases. The phylogeny of nifH is largely congruent with 16S rRNA phylogeny and, besides the cyanobacterial sequences, comprises a range of clusters representing presumably heterotrophic prokaryotes, including diverse Proteobacteria, Firmicutes, Spirochetes and Archaea (Zehr et al., 2003a). In recent years, various studies have suggested that such non-cyanobacterial prokaryotes must also be ecologically significant members of the oceanic diazotroph community, based on the fact that their nifH sequences are frequently detected or even prevalent in clone libraries or in libraries obtained from pyrosequencing of nifH amplicons (Riemann et al., 2010; Farnelid et al., 2011). Diazotroph communities appear to differ among surface waters of the open ocean, deep oceanic waters and the coastal ocean, reflecting different selective pressures acting on diazotrophs in these envi- ronments, and indicating that there are ecological niches specifically suited for non-cyanobacterial diazotrophs (Zehr et al., 2003a; Langlois et al., 2005). The presence and activity of presumably heterotrophic N 2-fixing bacteria in open ocean surface waters presents an enigma, since there are very low concentrations of suitable dissolved organic matter to fuel N2 fixation, and the surface oceans are saturated with oxygen, which inactivates nitrogenase, thereby inhibiting N2 fixation. Culture-independent approaches using quantitative PCR (qPCR) assays to enumerate nifH genes of non- Received 25 November, 2012; revised 26 April, 2013; accepted 5 May, 2013. *For correspondence. E-mail dbombar@ucsc.edu; Tel. (+1) 831 459 3128; Fax (+1) 831 459 4882. Environmental Microbiology Reports (2013) 5(5), 705–715 doi:10.1111/1758-2229.12070 © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology