Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation Wei Qina, Shady A. Aminb, Willm Martens-Habbenaa, Christopher B. Walkera, Hidetoshi Urakawac, Allan H. Devolb, Anitra E. Ingallsb, James W. Moffettd, E. Virginia Armbrustb, and David A. Stahla,1 aDepartment of Civil and Environmental Engineering and bSchool of Oceanography, University of Washington, Seattle, WA 98195; cDepartment of Marine and Ecological Sciences, Florida Gulf Coast University, Fort Myers, FL 33965; and dDepartment of Biological Sciences, University of Southern California, Los Angeles, CA 90089 Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved July 11, 2014 (received for review December 30, 2013) Ammonia-oxidizing archaea (AOA) are now implicated in exerting other abundant marine plankton (including Pelagibacter and significant control over the form and availability of reactive nitrogen Prochlorococcus) was made possible by genomic and biochemical species in marine environments. Detailed studies of specific meta- characterization of SCM1 (13, 14). More recent physiological bolic traits and physicochemical factors controlling their activities studies examining the copper requirements of SCM1 provided and distribution have not been well constrained in part due to the a framework to evaluate the significance of copper in controlling scarcity of isolated AOA strains. Here, we report the isolation of two the environmental distribution and activity of marine AOA (12). new coastal marine AOA, strains PS0 and HCA1. Comparison of the The capture of a greater representation of AOA environ- new strains to Nitrosopumilus maritimus strain SCM1, the only ma- mental diversity in pure culture should therefore serve to expand rine AOA in pure culture thus far, demonstrated distinct adaptations understanding of traits influencing their activity patterns in to pH, salinity, organic carbon, temperature, and light. Strain PS0 coastal and open marine systems. For example, a capacity to use sustained nearly 80% of ammonia oxidation activity at a pH as low fixed carbon and urea as alternative carbon and energy sources, as 5.9, indicating that coastal strains may be less sensitive to the respectively, has been suggested by tracer, metagenomic, and ongoing reduction in ocean pH. Notably, the two novel isolates are metatranscriptomic studies (25–33). The contribution of AOA to obligate mixotrophs that rely on uptake and assimilation of or- endogenously generated nitrate in marine surface waters, of ganic carbon compounds, suggesting a direct coupling between fundamental relevance to the source of nitrogen sustaining pri- chemolithotrophy and organic matter assimilation in marine food mary production in the photic zone, was suggested by relating webs. All three isolates showed only minor photoinhibition at 15 μE·m−2·s−1 and rapid recovery of ammonia oxidation in the nitrification activity and the archaeal amoA (the gene coding for the α-subunit of the ammonia monooxygenase) distribution dark, consistent with an AOA contribution to the primary nitrite – maximum and the plausibility of a diurnal cycle of archaeal ammo- patterns (7, 8, 34 36). A reported sensitivity of marine pop- nia oxidation activity in the euphotic zone. Together, these findings ulations to small reductions in pH, important to understanding highlight an unexpected adaptive capacity within closely related the possible impact of ongoing ocean acidification on the marine marine group I Archaea and provide new understanding of the nitrogen cycle, was inferred from a pH perturbation study (37). physiological basis of the remarkable ecological success reflected Thus, as was shown for studies of SCM1, the availability of ad- by their generally high abundance in marine environments. ditional AOA isolates will both inform inferences made in the field as well as provide a physiological basis to direct future marine ammonia-oxidizing archaea | ecophysiology | urea utilization field research. he discovery of ammonia-oxidizing archaea (AOA), some- Significance Ttimes constituting up to nearly 40% of marine microbial plankton, challenged the traditional view of microbial controls of Ammonia-oxidizing archaea (AOA) influence the form and nitrogen speciation in the ocean (1–5). The AOA are now gen- availability of nitrogen in marine environments and are a major erally recognized as the major drivers of nitrification in marine contributor to N2O release and plausible indirect source of environments (5–8). Their activities are of importance to trophic methane in the upper ocean. Thus, their sensitivity to ocean interactions that influence primary production and export of car- acidification and other physicochemical changes associated bon to the deep ocean, they are a known source of atmospheric with climate change has global significance. Here, we report on greenhouse gases (nitrous oxide and indirectly methane), and their the physiological response of marine AOA isolates to key en- high demand for copper may also alter food web dynamics (9–14). vironmental variables. Although reported as highly sensitive to Although enrichments of AOA strains belonging to groups reduction in ocean pH, we now show that some coastal marine I.1a and I.1b from a variety of marine and terrestrial environ- AOA can remain active with increasing acidification of the ments have been reported (15–22), no additional pure cultures of oceans. All AOA isolates assimilate fixed carbon and two are marine representatives have been described since the isolation of obligate mixotrophs, suggesting this globally significant as- Nitrosopumilus maritimus strain SCM1 (henceforth referred to as semblage serves a significant function in coupling chemolitho- SCM1) (23). However, many complex biological traits of signif- trophy with organic matter assimilation in marine food webs. icance to marine biogeochemistry and marine food webs cannot Author contributions: W.Q., S.A.A., W.M.-H., C.B.W., H.U., and D.A.S. designed research; be unambiguously established using metagenomic, metatran- W.Q., S.A.A., W.M.-H., C.B.W., and H.U. performed research; W.Q., S.A.A., and D.A.S. scriptomic studies and enrichment cultures. Initial understanding analyzed data; and W.Q., A.H.D., A.E.I., J.W.M., E.V.A., and D.A.S. wrote the paper. of the physiological basis for high AOA abundance in the marine The authors declare no conflict of interest. water column came from the demonstration that SCM1 is an This article is a PNAS Direct Submission. extreme oligotroph, having one of the highest affinities (low Ks) Data deposition: The sequences reported in this paper have been deposited in the GenBank for ammonia (here defined as combined ammonia/ammonium) database (accession nos. KF957663–KF957666). yet observed in pure culture (24). The discovery of a previously 1To whom correspondence should be addressed. Email: [email protected]. unidentified pathway for methylphosphonate synthesis, a plausi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ble source of methane in the upper ocean, in the AOA and 1073/pnas.1324115111/-/DCSupplemental. 12504–12509 | PNAS | August 26, 2014 | vol. 111 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1324115111 Downloaded by guest on October 2, 2021 We now report the isolation of two novel coastal marine AOA A B strains from the Puget Sound estuary system in Washington, signif- 500 50 500 50 - icantly expanding the ecotypic diversity of marine Thaumarchaeota NO - + NO2 + 2 NH4 400 40 Cell number [10 400 NH4 40 Cell number [10 M] available in pure culture. Despite relatively close phylogenetic M] relationships, the three marine isolates comprise physiologically 300 30 300 30 Cells distinct ecotypes of AOA, varying in their capacity to use different Cells 200 20 200 20 6 6 ml carbon and energy sources, and in their tolerance to changes in pH, ml -1 -1 ] salinity, and light. These distinctive differences are directly relevant ] Ammonium, nitrite [ Ammonium, Ammonium, nitrite [ Ammonium, 100 10 100 10 to their possible contribution to nitrification in different marine environments—including lower salinity coastal regions, the photic 0 0 0 0 0 50 100 150 200 250 0 100 200 300 400 500 zone of the upper water column, and the increasing acidification of Time [h] Time [h] ocean waters associated with climate change. C D Results Enrichment and Isolation of Marine AOA. Enrichment cultures were initiated from Puget Sound main basin near surface (47.55 N, 122.28 W) and 50 m water from the Puget Sound Regional Synthesis Model (PRISM) Station P10 (47.91 N, 122.62 W) in Hood Canal. Our previous molecular surveys showed these coastal waters to be dominated by AOA, relative to ammonia- oxidizing bacteria (AOB) (8, 38, 39). Predominance of AOA was consistent with generally submicromolar concentrations of am- monia at these stations, sufficient to support AOA such as SCM1 Fig. 1. Growth and morphology of strains HCA1 and PS0. Correlation be- but not known AOB (24). Enrichment conditions were designed tween ammonia oxidation and growth of HCA1 (A)andPS0(B) in an arti- μ + μ α to simulate the low substrate availability of these marine systems. ficial seawater medium containing 500 MNH4 and 100 M -ketoglutaric μ acid. Transmission electron micrographs of negative-stained cells of HCA1 The growth medium was supplemented with 2 MNH4Cl, and (C) and PS0 (D). (Scale bars: 200 nm.) cultures were incubated at 15 °C (SI Materials and Methods). Once the enrichments showed stable activity, they were transferred to artificial seawater medium supplemented with in situ cell-specific rates of a natural marine community (0.2–15 μ μ α + 2 MNH4Cl and 100 M -ketoglutaric acid. Earlier studies of fmol of NH4 per cell per d) (6). strain SCM1 had shown that only a few central metabolites, in- cluding α-ketoglutaric acid, stimulated growth (SI Materials and Morphology and Phylogeny. The morphologies of strains HCA1 Methods). Highly enriched AOA cultures were obtained follow- and PS0, as characterized by transmission electron microscopy ing ∼2 y of consecutive transfer of 10% (vol/vol) late–exponen- (Fig.