The ISME Journal (2012) 6, 951–960 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE Denitrification likely catalyzed by endobionts in an allogromiid foraminifer Joan M Bernhard1, Virginia P Edgcomb1, Karen L Casciotti2,4, Matthew R McIlvin2 and David J Beaudoin3 1Geology and Geophysics Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA; 2Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA and 3Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA Nitrogen can be a limiting macronutrient for carbon uptake by the marine biosphere. The process of denitrification (conversion of nitrate to gaseous compounds, including N2 (nitrogen gas)) removes bioavailable nitrogen, particularly in marine sediments, making it a key factor in the marine nitrogen budget. Benthic foraminifera reportedly perform complete denitrification, a process previously considered nearly exclusively performed by bacteria and archaea. If the ability to denitrify is widespread among these diverse and abundant protists, a paradigm shift is required for biogeochemistry and marine microbial ecology. However, to date, the mechanisms of foraminiferal denitrification are unclear, and it is possible that the ability to perform complete denitrification is because of the symbiont metabolism in some foraminiferal species. Using sequence analysis and GeneFISH, we show that for a symbiont-bearing foraminifer, the potential for denitrification resides in the endobionts. Results also identify the endobionts as denitrifying pseudomonads and show that the allogromiid accumulates nitrate intracellularly, presumably for use in denitrification. Endobionts have been observed within many foraminiferal species, and in the case of associations with denitrifying bacteria, may provide fitness for survival in anoxic conditions. These associations may have been a driving force for early foraminiferal diversification, which is thought to have occurred in the Neoproterozoic era when anoxia was widespread. The ISME Journal (2012) 6, 951–960; doi:10.1038/ismej.2011.171; published online 1 December 2011 Subject Category: microbe–microbe and microbe–host interactions Keywords: denitrification; GeneFISH; nirK; marine sediment; Pseudomonas; symbiosis Introduction foraminiferal species from a wide taxonomic range have been shown to store nitrate, and of the ten Over the past decade, our understanding of the foraminiferal species analyzed for denitrification nitrogen cycle has changed drastically (Francis rates, nine perform complete denitrification. et al., 2007) with the discovery of anaerobic ammo- However, our understanding of the mechanisms of nium oxidation (anammox, Dalsgaard et al., 2005), denitrification in these protists is incomplete. archaeal ammonia oxidation (Ko¨nneke et al., 2005) We addressed uncertainties about foraminiferal and the ability of foraminiferal eukaryotes to perform denitrification in a symbiont-bearing allogromiid complete denitrification (Risgaard-Petersen et al., foraminifer. Although allogromiid foraminifera, 2006). The first report of benthic foraminifera having which are tectinous and unilocular, differ in the ability to perform complete denitrification was morphology and test (shell) chemistry from the exciting because denitrification was thought to be a multilocular calcareous Globobulimina pseudospi- process facilitated only by prokaryotes and some nescens, which was the first foraminiferal species fungi. Additional studies suggest that the ability of determined to perform denitrification (Risgaard- foraminifera to denitrify is widespread (Høgslund Petersen et al., 2006), the species live in similar et al., 2008; Pin˜a-Ochoa et al., 2010). Dozens of habitats. The SBB (Santa Barbara Basin) allogromiid lives in relatively high abundance in sediments of Correspondence: JM Bernhard, Geology and Geophysics Depart- the deepest part of the SBB (Bernhard et al., 2006), ment, Woods Hole Oceanographic Institution, Woods Hole, MA and is the only allogromiid common in this part of 02543, USA. the basin where denitrification might be expected E-mail: [email protected] because bottom-water oxygen concentrations in this 4Current address: Stanford University, Department of Environ- mental Earth System Science, Stanford, CA 94305, USA. area can be very low (typically o2 mM) (Bernhard Received 15 August 2011; revised 30 September 2011; accepted et al., 1997) or undetectable (Bernhard et al., 2006). 11 October 2011; published online 1 December 2011 At times, sulfide concentrations can be considerable Denitrification in an allogromiid foraminifer JM Bernhard et al 952 in this area (Bernhard et al., 2003). The species was Materials and methods selected because of a number of its attributes, including its ecology, abundant endobionts and Sample collection copious large cytoplasmic vacuoles (Bernhard Samples were collected from water depths of 580– et al., 2006) (Figure 1). The abundant endobionts 590 m in SBB, which is a silled basin with restricted of this SBB allogromiid appear to be one morpho- water circulation located off Southern California, USA 1 1 0 type in the many specimens examined previously (centered on 34 13.5N, 120 02 W(Reimerset al., 1990; (Bernhard et al., 2006). The copious large cytoplas- Reimers et al., 1996). Sediments were collected on five mic vacuoles in this allogromiid are easily observed, occasions (9/07, 6/08, 10/08, 6/09 and 4/10) with a and were noted in both fixed and live specimens in Soutar boxcorer (Ocean Instruments, San Diego, CA, a prior cell-ultrastructural study (Bernhard et al., USA), from which various samples were obtained. 2006). Although this foraminiferal species is not Those intended as sources for live material were formally described, it is morphologically distinct placed along with bottom water in tightly sealed and its small subunit ribosomal RNA gene sequence high-density polyethylene bottles, kept chilled and (GenBank accession number AY818728) was found transported to our lab where they were maintained 1 to group consistently in the basal foraminiferal at 7 C, which is near ambient temperature. lineage ‘Clade L’ (Bernhard et al., 2006). In the earlier study of this SBB allogromiid, the Nitrate content and isotopic composition identity of the endobiont was unknown but was To determine intracellular nitrate concentrations, hypothesized to be a sulfur-oxidizing bacterium live specimens were isolated from sediments by (Bernhard et al., 2006). The reports of denitrification gently sieving over a 90-mm screen using chilled in foraminifera spawned two new hypotheses, bottom water as soon as possible (within a few days) however: (1) that the allogromiid’s large cytoplasmic after returning to the laboratory, rinsed twice in vacuoles contained nitrate and (2) that the endo- 0.2 mm-filter, nitrate-free seawater, measured for length bionts were denitrifiers. Using a variety of geochem- and diameter using an ocular micrometer mounted ical and molecular approaches, we address these on a Nikon SMZ 2B stereomicroscope (Nikon, hypotheses and consider the results with respect to New York, NY, USA), and individually air dried in chemocline biogeochemistry and ecology, as well as 0.2 ml acid-cleaned polypropylene tubes. Nitrate early foraminiferal diversification. content and isotopic composition were measured Figure 1 Transmission electron microscopy micrographs of the SBB allogromiid. (a) Low magnification view showing abundant vacuoles (v) in the endoplasm vs ectoplasm; e, environment outside foraminifer. (b) View of coccoid endobionts showing their tendency to form short chains (*). Scales: a ¼ 5 mm; b ¼ 1 mm. The ISME Journal Denitrification in an allogromiid foraminifer JM Bernhard et al 953 using isotope ratio mass spectrometry with the nirK and Pseudomonas stutzeri for nirS. Allogromiid denitrifier method (Sigman et al., 2001), which specimens were also screened for sulfur oxidation À relies on conversion of NO3 (nitrate) to N2O (nitrous and sulfate reduction genes soxB and dsrAB, oxide) and sensitive detection of N2O via isotope respectively, with Thiomicrospira species strain ratio mass spectrometry. Air-dried individuals were L-12 and Desulfovibrio vulgaris as positive controls, dissolved in 100 ml acetic acid, and replicate 50 ml respectively, using established primers and proto- aliquots were analyzed for nitrate content against cols (Petri et al., 2001). Dissimilatory (bi)sulfite gene low-level (0.05–1.0 nmol) nitrate standards using the amplifications were run using the DSR1Fmix and peak area of the major ion beam (m/z ¼ 44). DSR4Rmix primer sets (Loy et al., 2004). Intracellular nitrate concentrations were determined PCR products from all positive amplifications were using recorded dimensions and volume calculations gel-purified using the Qiaquick Gel Extraction Kit 1 2 for a cylinder prolate spheroid (p[2W] L, where (Qiagen) and cloned into the pCR4 vector in the W ¼ width and L ¼ length). When nitrate content TOPO TA cloning kit (Invitrogen, Carlsbad, CA, was sufficient (40.5 nmol), the d15N of the nitrate USA), according to the manufacturer’s instructions 15 15 14 15 14 (d NNO3 ¼ (( N/ N)NO3/( N/ N)AIRÀ1) Â 1000) was (for 357F/1492R, four separate amplification pro- 15 determined for the individual. d NNO3 was normal- ducts were pooled before cloning). Selected clones ized to the AIR reference scale by the analysis of (74 from 357F/1542R and 16–24 clones from