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Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences

Nature Microbiology volume 9, pages 524–536 (2024)Cite this article

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Abstract

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (β-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while β-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

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Fig. 1: Growth curves and maximum specific growth rate (µmax).
Fig. 2: 15N stable isotope tracking the ammonia and urea utilization in N. lacus.
Fig. 3: Michaelis–Menten kinetic constants of ammonia- and urea-dependent oxidation.
Fig. 4: Transcriptional response of AOM species to substrate switch from a single N source to a mixture of ammonia and urea.

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Data availability

All data are available in the main text or the supplementary materials. RNA-seq data in this study have been deposited in the NCBI GEO database under BioProject ID PRJNA896729. All nitrifier genomes in this study were downloaded from the public NCBI RefSeq database (https://www.ncbi.nlm.nih.gov/refseq/). Source data are provided with this paper.

Change history

  • 28 March 2024

    In the version of the article initially published, Supplementary Tables 1 and 2 were switched with Source Data Tables 1 and 2. This has now been corrected.

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Acknowledgements

We thank Z. Perry, B. Quan and T. Tubbs for technical assistance in growth experiments and S. Oleynik for maintaining the mass spectrometer for stable isotope analysis. N. inopinata was generously provided by H. Daims, University of Vienna. N. piranensis was generously provided by A. Santoro and B. Bayer, University of California, Santa Barbara. N. multiformis was kindly provided by J. Norton, Utah State University. N. ureae was kindly provided by A. Pommerening-Röser, University of Hamburg. This work was supported by National Natural Science Foundation of China grants 42277304 and 41977056 (to B.W.) and 42276117 (Y. Zheng). This work was also supported by the startup funding of the University of Oklahoma (to W.Q.). Work was also funded by the US Department of Energy’s Office of Science, Division of Biological and Environmental Research Program (Grant DE-SC0020356 to M.-K.H.W., X.M., D.A.S., C.P., Z.F., B.A. and S.P.W.) and by the Defense Advanced Research Projects Agency (Contract Number HR0011-17-2-0064 to M.-K.H.W., D.A.S. and S.P.W.). Funding was also provided by Florida Agricultural Experiment Station Hatch project FLA-FTL-005680, UF IFAS Early Career Award and USDA NIFA award number 2022-67019-36501 (to W.M.-H.). Part of this work was performed at the Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. The participation of X.W. and B.B.W. was supported by Simons Foundation grant 675459 to B.B.W. X.S. was supported by a G. Evelyn Hutchinson postdoctoral fellowship from Yale Institute for Biospheric Studies at Yale University. H.U. was supported by National Science Foundation grant DEB-1664052.

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Author notes

  1. These authors contributed equally: Wei Qin, Stephany P. Wei, Yue Zheng, Eunkyung Choi.

Authors and Affiliations

  1. School of Biological Sciences, Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA

    Wei Qin, Xiangpeng Li, Hanyan Li, Lei Hou, Qing Tao, Wyatt W. Chlouber, Michael Wells, Long Ngo, Xuanyu Tao, Dongyu Wang, Chongle Pan & Jizhong Zhou

  2. Department of Civil and Environmental Engineering, University of Washington, Seattle, WA, USA

    Wei Qin, Stephany P. Wei, Britt Abrahamson, Zachary Flinkstrom, Kristopher A. Hunt, David A. Stahl & Mari-Karoliina H. Winkler

  3. State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian, China

    Yue Zheng, Lei Hou & Dazhi Wang

  4. Department of Microbiology and Cell Science, Fort Lauderdale Research and Education Center, University of Florida, Davie, FL, USA

    Eunkyung Choi & Willm Martens-Habbena

  5. Lawrence Livermore National Laboratory, Livermore, CA, USA

    Juliet Johnston, Peter K. Weber & Xavier Mayali

  6. Department of Geosciences, Princeton University, Princeton, NJ, USA

    Xianhui Wan & Bess B. Ward

  7. College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, China

    Baozhan Wang & Jiandong Jiang

  8. Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA

    Xin Sun

  9. Department of Ecology and Environmental Studies, Florida Gulf Coast University, Fort Myers, FL, USA

    Hidetoshi Urakawa

  10. State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China

    Xiaoyuan Yan

  11. State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian, China

    Yao Zhang

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  1. Wei Qin
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Contributions

W.Q., D.A.S., B.B.W., X.M., W.M.-H. and M.-K.H.W. designed the experiments. W.Q., S.P.W., Y. Zheng, E.C., X.L., J. Johnston, X.W., B.A. and Z.F. performed the experiments and analysed data with input from B.W., H.L., L.H., Q.T., W.W.C., X.S., M.W., L.N., K.A.H., H.U., X.T., Dongyu Wang, X.Y., Dazhi Wang, C.P., P.K.W., J. Jiang, Y. Zhang and J.Z. W.Q., S.P.W., D.A.S., W.M.-H. and M.-K.H.W. wrote the paper, with contributions and approval from all other authors.

Corresponding authors

Correspondence to Wei Qin, Willm Martens-Habbena or Mari-Karoliina H. Winkler.

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The authors declare no competing interests.

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Nature Microbiology thanks Marc Strous and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The phylogenetic pattern of ammonia and urea uptake and utilization genes in AOA (a), AOB (b), and comammox (c).

The phylogenomic trees were constructed based on concatenated sequences of 122 and 120 single-copy marker genes shared among archaea and bacteria, respectively73. Confidence values are based on 1000 bootstrap replications. Scale bars represent 20%, 10%, and 5% estimated sequence divergence for AOA, AOB, and comammox phylogenomic trees, respectively. The estimated completeness of each genome is displayed next to the taxon name. The gene copy number is indicated by the darkness of the color in the cycles. *Note that the genes involved in the ATP-dependent urea hydrolysis pathway were transcribed at very low levels during urea consumption by β-AOB N. lacus, suggesting a different function of these conserved enzymes in β-AOB. ut: DUR3-type urea transporter; urt: ATP-dependent urea ABC transporter; utp: yut-type urea transporter; nikR: nickel-responsive transcriptional regulator; ureA–C: urease; ureD–H, J: urease accessory protein; uc: putative urea carboxylase; ucp12: putative urea carboxylase accessory protein; amt: Amt-type ammonium transporter; Rh-type amt, Rhesus-type ammonia transporter; glnB/K: nitrogen regulatory PII protein; glnL: nitrogen regulation sensor histidine kinase GlnL; glnG: nitrogen regulation response regulator GlnG; ntrX: nitrogen regulation response regulator NtrX; ntrY: nitrogen regulation sensor histidine kinase NtrY; glnD: bifunctional PII protein uridylyltransferase/uridylyl-removing enzyme; amo: ammonia monooxygenase; gudB: glutamate dehydrogenase; GDH2: glutamate dehydrogenase; GLUD1_2: glutamate dehydrogenase (NAD(P)+); gdhA: glutamate dehydrogenase (NADP+); glt: NADPH-dependent glutamate synthase; glnA: glutamine synthetase; sbt: bicarbonate transporter; ca: carbonic anhydrase; SAP: short chain amide/urea porin.

Extended Data Fig. 2 Schematic illustration of the genomic regions of the AOM species tested in this study containing genes for ammonia and urea uptake and utilization.

Gene functions are indicated by color: Grey, ammonia oxidation and assimilation; green, urea transporters; purple, putative outer membrane short chain amide/urea porins; orange, urease and accessory proteins; yellow, nitrogen regulatory PII proteins and Nickel-responsive regulator; blue, bicarbonate transporter and carbonic anhydrase; red, putative allophanate hydrolase, urea carboxylase, and accessory proteins. Genes are drawn to scale, and the scale bar corresponds to 1000 bp sequence length. Gene full name/abbreviation pairs are the same as shown in Extended Data Fig. 1 and Supplementary Table 3.

Extended Data Fig. 3 15N stable isotope tracking the nitrite/nitrate production and ammonia secretion in N. lacus, N.oceani and N. inopinata.

Tracking nitrite/nitrate production and ammonia secretion for the incubations containing 15N-ammonia or 15N-urea. (a and b) N. lacus, (c and d) N. oceani, and (e and f) N. inopinata (n = 3 or 4 biologically independent incubations).

Extended Data Fig. 4 Summary of N assimilation results based on NanoSIMS.

(a) Schematic representations of ammonia and urea consumption profiles and indication of time points for cell collection. (b) % new N incorporation (average ± standard deviation of all analyzed cells) from ammonia and urea. (c) Example NanoSIMS image (scale bar = 5 µm) where top row shows preference for ammonia (images of the soil AOA N. viennensis cells) and bottom shows preference for urea (images of the β-AOB N. lacus cells). Over 15 NanoSIMS images were collected for each AOM species at each time point. See Supplementary Figs. 4 and Extended Data Fig. 5 for growth curves with the time points indicated for each organism and the NanoSIMS images for other investigated AOM species, respectively. The NanoSIMS analysis software output data, which were processed to calculate % incorporation from substrates, are provided in Source Data Table 2.

Extended Data Fig. 5 Example NanoSIMS images for the marine AOA N. piranensis, comammox N. inopinata, and γ-AOB N. oceani.

Top and bottom rows represent the NanoSIMS images taken at T1 for labeled urea and labeled ammona incubations, respectively. Over 15 NanoSIMS images were collected for each AOM species at each time point. The NanoSIMS analysis software output data, which were processed to calculate % incorporation from substrates, are provided in Source Data Table 2.

Extended Data Fig. 6 Percent of N incorporation from ammonia-N or urea-N determined by nanoSIMS for single cells of (a) N. piranensis (marine AOA), (b) N. viennensis (soil AOA), (c) N. inopinata (comammox), (d) N. lacus (β-AOB), and (e) N. oceani (γ-AOB).

N incorporation data were obtained in multiple filters harvesting cells from biological triplicate incubations (n = 3). The box plots represent the median as well as the 25% and 75% interquartile ranges. The whiskers represent 1.5× the interquartile ranges.

Extended Data Fig. 7 Percent of C incorporation from bicarbonate-C or urea-C determined by NanoSIMS for single cells of (a) N. piranensis (marine AOA), (b) N. viennensis (soil AOA), (c) N. inopinata (comammox), (d) N. lacus (β-AOB), and (e) N. oceani (γ-AOB).

Note that Y-axes are in different scales for bicarbonate-C (red) and urea-C (blue) incorporation. C incorporation data were obtained in multiple filters harvesting cells from biological triplicate incubations (n = 3). The box plots represent the median as well as the 25% and 75% interquartile ranges. The whiskers represent 1.5× the interquartile ranges.

Extended Data Fig. 8 Ammonia- or urea-dependent substrate oxidation kinetics of N. viennensis, N. piranensis, N. inopinata, N. lacus, and N. multiformis.

a–e, Ammonia-grown strains, and f–o, Urea-grown strains. Each graph represents a single trace of experimental replicates. Oxygen uptake rates were determined by a microrespirometry (MR) measurement upon substrate addition. Kinetic constants were obtained by fitting a Michaelis-Menten equation (red line) to total ammonia or urea and oxidation rates.

Extended Data Fig. 9 15N stable isotope tracking the ammonia and urea utilization in N. oceani and N. inopinata.

The 15N percentage of accumulated nitrite in the medium was measured for 15N-NH3 treated incubations (a and b) and 15N-urea treated incubations (c and d) and the contribution of ammonia and urea to nitrite production was calculated for (a, c) N. oceani and to nitrite and nitrate production for (b, d) N. inopinata (n = 3 biologically independent incubations). Error bars represent the standard deviations of the biological triplicate incubations. Total concentrations of N species are plotted in light grey on the secondary y axis for background reference.

Extended Data Fig. 10 Instantaneous measurement of oxygen uptake with N. viennensis, N. piranensis, and N. inopinata.

a–c, Ammonia-grown strains, and d–f, urea-grown strains. Arrows: the injection of ammonia or urea into the chamber via an injection port using a 2 mL syringe.

Supplementary information

Supplementary Information

Supplementary Discussion, Figs. 1–7, Tables 1–3 (table titles only), Table 4, and Source Data Tables 1 and 2 (table titles only).

Reporting Summary

Supplementary Table 1

P values from two-tailed t-test (α = 0.05) pairing the maximum specific growth rate between different incubation experiments. Values <0.05 are highlighted in green. Letters without square brackets indicate treatment with different N substrates: A, incubation with ammonia only; A + U, incubation with ammonia + urea; U, incubation with urea only. Letters in square brackets indicate the substrate being utilized when calculated for the µmax: A, when using ammonia; U, when using urea; A + U, when using ammonia and urea together. *Inoculation with 1% urea-grown maintenance culture at mid-exponential phase; all other incubations were inoculated with 1% ammonia-grown maintenance culture.

Supplementary Table 2

The summarized ammonia- and urea-dependent oxidation kinetics parameters of N. piranensis, N. viennensis, N. inopinata, N. lacus and N. multiformis.

Supplementary Table 3

The summarized transcription data of ammonia and urea uptake and utilization gene in AMO species.

Source data

Source Data Table 1

Differential transcription of all AMO species genes in response to ammonia and urea additions.

Source Data Table 2

NanoSIMS analysis output (‘Raw’) and processed data (‘Data’) for calculating substrate incorporation percentages.

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Qin, W., Wei, S.P., Zheng, Y. et al. Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences. Nat Microbiol 9, 524–536 (2024). https://doi.org/10.1038/s41564-023-01593-7

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