Skip to main content
SpringerLink
Log in

The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state.

Graphic abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Le Treut H, Somerville R, Cubasch U, Ding Y, Mauritzen C, Mokssit A, Peterson T and Prather M (2007) In: Baede A, Griggs D (eds) Climate Change 2007: The Physical Science Basis Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge

  2. Fowler D, Coyle M, Skiba U, Sutton MA, Cape JN, Reis S, Sheppard LJ, Jenkins A, Grizzetti B, Galloway JN, Vitousek P, Leach A, Bouwman AF, Butterbach-Bahl K, Dentener F, Stevenson D, Amann M, Voss M (2013) The global nitrogen cycle in the twenty-first century. Philos Trans R Soc Lond B Biol Sci 368:20130164

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Mosier A, Carolien K, Cindy N, Oene O, Sybil S (1998) Closing the global N2O budget: nitrous oxide emissions through the agricultural nitrogen cycle inventory methodology. Nutr Cycl Agroecosyst 52:225–248

    Article  CAS  Google Scholar 

  4. Lassey K, Harvey M (2007) Nitrous oxide: the serious side of laughing gas. Water Atmos 15(2):10–11

  5. Thomson AJ, Giannopoulos G, Pretty J, Baggs EM, Richardson DJ (2012) Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Philos Trans R Soc Lond B Biol Sci 367:1157–1168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Voss M, Bange HW, Dippner JW, Middelburg JJ, Montoya JP, Ward B (2013) The marine nitrogen cycle: recent discoveries, uncertainties and the potential relevance of climate change. Philos Trans R Soc Lond B Biol Sci 368:20130121

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Richardson D, Felgate H, Watmough N, Thomson A, Baggs E (2009) Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle—could enzymic regulation hold the key? Trends Biotechnol 27:388–397

    Article  CAS  PubMed  Google Scholar 

  8. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Philippot L (2002) Denitrifying genes in bacterial and archaeal genomes. Biochim Biophys Acta 1577:355–376

    Article  CAS  PubMed  Google Scholar 

  10. Zumft WG, Kroneck PM (2007) Respiratory transformation of nitrous oxide (N2O) to dinitrogen by bacteria and archaea. Adv Microb Physiol 52:107–227

    Article  CAS  PubMed  Google Scholar 

  11. Pauleta SR, Carepo MSP, Moura I (2019) Source and reduction of nitrous oxide. Coord Chem Rev 387:436–449

    Article  CAS  Google Scholar 

  12. Hallin S, Philippot L, Loffler FE, Sanford RA, Jones CM (2018) Genomics and ecology of novel N2O-reducing microorganisms. Trends Microbiol 26:43–55

    Article  CAS  PubMed  Google Scholar 

  13. Scott RA, Zumft WG, Coyle CL, Dooley DM (1989) Pseudomonas stutzeri N2O reductase contains CuA-type sites. Proc Natl Acad Sci USA 86:4082–4086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Brown K, Djinovic-Carugo K, Haltia T, Cabrito I, Saraste M, Moura JJG, Moura I, Tegoni M, Cambillau C (2000) Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur. J Biol Chem 275:41133–41136

    Article  CAS  PubMed  Google Scholar 

  15. Simon J, Einsle O, Kroneck PM, Zumft WG (2004) The unprecedented nos gene cluster of Wolinella succinogenes encodes a novel respiratory electron transfer pathway to cytochrome c nitrous oxide reductase. FEBS Lett 569:7–12

    Article  CAS  PubMed  Google Scholar 

  16. Johnston EM, Dell'Acqua S, Ramos S, Pauleta SR, Moura I, Solomon EI (2014) Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase. J Am Chem Soc 136:614–617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dell'Acqua S, Pauleta SR, Moura JJ, Moura I (2012) Biochemical characterization of the purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase. Philos Trans R Soc Lond B Biol Sci 367:1204–1212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rasmussen T, Berks BC, Butt JN, Thomson AJ (2002) Multiple forms of the catalytic centre, CuZ, in the enzyme nitrous oxide reductase from Paracoccus pantotrophus. Biochem J 364:807–815

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fujita K, Chan JM, Ja B, Alvarez ML, Dooley DM (2007) Anaerobic purification, characterization and preliminary mechanistic study of recombinant nitrous oxide reductase from Achromobacter cycloclastes. J Inorg Biochem 101:1836–1844

    Article  CAS  PubMed  Google Scholar 

  20. Pomowski A, Zumft WG, Kroneck PMH, Einsle O (2011) N2O binding at a 4Cu:2S copper-sulphur cluster in nitrous oxide reductase. Nature 477:234–237

    Article  CAS  PubMed  Google Scholar 

  21. Prudêncio M, Pereira AS, Tavares P, Besson S, Cabrito I, Brown K, Samyn B, Devreese B, Van Beeumen J, Rusnak F, Fauque G, Moura JJ, Tegoni M, Cambillau C, Moura I (2000) Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617. Biochemistry 39:3899–3907

    Article  PubMed  CAS  Google Scholar 

  22. Dell'acqua S, Pauleta SR, Monzani E, Pereira AS, Casella L, Moura JJ, Moura I (2008) Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies. Biochemistry 47:10852–10862

    Article  CAS  PubMed  Google Scholar 

  23. Ghosh S, Gorelsky SI, Chen P, Cabrito I, Moura JJG, Moura I, Solomon EI (2003) Activation of N2O reduction by the fully reduced micro4-sulfide bridged tetranuclear CuZ cluster in nitrous oxide reductase. J Am Chem Soc 125:15708–15709

    Article  CAS  PubMed  Google Scholar 

  24. Chan JM, Bollinger J, Grewell CL, Dooley DM (2004) Reductively activated nitrous oxide reductase reacts directly with substrate. J Am Chem Soc 126:3030–3031

    Article  CAS  PubMed  Google Scholar 

  25. Carreira C, Dos Santos MMC, Pauleta SR, Moura I (2020) Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus—an electrochemical study. Bioelectrochemistry 133:107483

    Article  CAS  PubMed  Google Scholar 

  26. Wunsch P, Zumft WG (2005) Functional domains of nosr, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration. J Bacteriol 187:1992–2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wunsch P, Körner H, Neese F, van Spanning RJ, Kroneck PM, Zumft WG (2005) NosX function connects to nitrous oxide (N2O) reduction by affecting the Cu(Z) center of NosZ and its activity in vivo. FEBS Lett 579:4605–4609

    Article  CAS  PubMed  Google Scholar 

  28. Zhang L, Trncik C, Andrade SLA, Einsle O (2017) The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction. Biochim Biophys Acta 1858:95–102

    Article  CAS  Google Scholar 

  29. Wallenstein MD, Myrold DD, Firestone M, Voytek M (2006) Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods. Ecol Appl 16:2143–2152

    Article  PubMed  Google Scholar 

  30. Sullivan MJ, Gates AJ, Appia-Ayme C, Rowley G, Richardson DJ (2013) Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism. Proc Natl Acad Sci USA 110:19926–19931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Smid AE, Beauchamp EG (1976) Effects of temperature and organic matter on denitrification in soil. Can J Soil Sci 56:385–391

    Article  CAS  Google Scholar 

  32. Saggar S, Jha N, Deslippe J, Bolan NS, Luo J, Giltrap DL, Kim DG, Zaman M, Tillman RW (2013) Denitrification and N2O:N2 production in temperate grasslands: processes, measurements, modelling and mitigating negative impacts. Sci Total Environ 465:173–195

    Article  CAS  PubMed  Google Scholar 

  33. Bergaust L, van Spanning RJM, Frostegard A, Bakken LR (2012) Expression of nitrous oxide reductase in Paracoccus denitrificans is regulated by oxygen and nitric oxide through FnrP and NNR. Microbiology 158:826–834

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hassan J, Qu Z, Bergaust LL, Bakken LR (2016) Transient accumulation of NO2− and N2O during denitrification explained by assuming cell diversification by stochastic transcription of denitrification genes. PLoS Comput Biol 12:e1004621

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Blum JM, Su Q, Ma Y, Valverde-Perez B, Domingo-Felez C, Jensen MM, Smets BF (2018) The pH dependency of N-converting enzymatic processes, pathways and microbes: effect on net N2O production. Environ Microbiol 20:1623–1640

    Article  PubMed  CAS  Google Scholar 

  36. Liu B, Frostegard A, Bakken LR (2014) Impaired reduction of N2O to N2 in acid soils is due to a posttranscriptional interference with the expression of nosZ. MBio 5:e01383–e11314

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Liu B, Mørkved PT, Frostegård A, Bakken LR (2010) Denitrification gene pools, transcription and kinetics of NO, N2O and N2 production as affected by soil pH. FEMS Microbiol Ecol 72:407–417

    Article  CAS  PubMed  Google Scholar 

  38. Imek M, Cooper JE (2002) The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years. Eur J Soil Sci 53:345–354

    Article  Google Scholar 

  39. Wan R, Wang L, Chen Y, Zheng X, Su Y, Tao X (2018) Insight into a direct carbon dioxide effect on denitrification and denitrifying bacterial communities in estuarine sediment. Sci Total Environ 643:1074–1083

    Article  CAS  PubMed  Google Scholar 

  40. Spiro S (2012) Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors. Philos Trans R Soc Lond B Biol Sci 367:1213–1225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Torres MJ, Simon J, Rowley G, Bedmar EJ, Richardson DJ, Gates AJ, Delgado MJ (2016) Nitrous oxide metabolism in nitrate-reducing bacteria: physiology and regulatory mechanisms. Adv Microb Physiol 68:353–432

    Article  CAS  PubMed  Google Scholar 

  42. Bergaust L, Mao Y, Bakken LR, Frostegård A (2010) Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrous oxide reductase in Paracoccus denitrificans. Appl Environ Microbiol 76:6387–6396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Alves T, Besson S, Duarte LC, Pettigrew GW, Girio FM, Devreese B, Vandenberghe I, Van Beeumen J, Fauque G, Moura I (1999) A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization. Biochim Biophys Acta 1434:248–259

    Article  CAS  PubMed  Google Scholar 

  44. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40:e115–e115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gomes JP, Hsia RC, Mead S, Borrego MJ, Dean D (2005) Immunoreactivity and differential developmental expression of known and putative Chlamydia trachomatis membrane proteins for biologically variant serovars representing distinct disease groups. Microbes Infect 7:410–420

    Article  CAS  PubMed  Google Scholar 

  46. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Article  CAS  PubMed  Google Scholar 

  47. Hanna PM, Tamilarasan R, McMillin DR (1988) Cu(I) analysis of blue copper proteins. Biochem J 256:1001–1004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kristjansson JK, Hollocher TC (1980) First practical assay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization. J Biol Chem 255:704–707

    Article  CAS  PubMed  Google Scholar 

  49. Carreira C, Mestre O, Nunes RF, Moura I, Pauleta SR (2018) Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes. PeerJ 6:e5603

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Bennett SP, Soriano-Laguna MJ, Bradley JM, Svistunenko DA, Richardson DJ, Gates AJ, Le Brun NE (2019) NosL is a dedicated copper chaperone for assembly of the CuZ center of nitrous oxide reductase. Chem Sci 10:4985–4993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bertsova YV, Fadeeva MS, Kostyrko VA, Serebryakova MV, Baykov AA, Bogachev AV (2013) Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins. J Biol Chem 288:14276–14286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Pauleta SR, Moura I (2017) Nitrous oxide reductase (CuZ and CuA). In: Johnson MK, Scott RA (eds) Encyclopedia of inorganic and bioinorganic chemistry; Vol. Metalloprotein active site Assembly. Wiley

  53. Carreira C, Pauleta SR, Moura I (2017) The catalytic cycle of nitrous oxide reductase—the enzyme that catalyzes the last step of denitrification. J Inorg Biochem 177:423–434

    Article  CAS  PubMed  Google Scholar 

  54. Ghosh S, Gorelsky SI, George DS, Chan JM, Cabrito I, Dooley DM, Moura JJG, Moura I, Solomon EI (2007) Spectroscopic, computational, and kinetic studies of the mu4-sulfide-bridged tetranuclear CuZ cluster in N2O reductase: pH effect on the edge ligand and its contribution to reactivity. J Am Chem Soc 129:3955–3965

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hahnke SM, Moosmann P, Erb TJ, Strous M (2014) An improved medium for the anaerobic growth of Paracoccus denitrificans Pd1222. Front Microbiol 5:18

    Article  PubMed  PubMed Central  Google Scholar 

  56. Baumann B, van der Meer JR, Snozzi M, Zehnder AJ (1997) Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH. Anton Leeuw Int J G 72:183–189

    Article  CAS  Google Scholar 

  57. Hartop KR, Sullivan MJ, Giannopoulos G, Gates AJ, Bond PL, Yuan Z, Clarke TA, Rowley G, Richardson DJ (2017) The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans. Water Res 113:207–214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Almeida JS, Julio SM, Reis MA, Carrondo MJ (1995) Nitrite inhibition of denitrification by Pseudomonas fluorescens. Biotechnol Bioeng 46:194–201

    Article  CAS  PubMed  Google Scholar 

  59. Schreiber F, Wunderlin P, Udert KM, Wells GF (2012) Nitric oxide and nitrous oxide turnover in natural and engineered microbial communities: biological pathways, chemical reactions, and novel technologies. Front Microbiol 3:372

    Article  PubMed  PubMed Central  Google Scholar 

  60. Bueno E, Mania D, Frostegard A, Bedmar EJ, Bakken LR, Delgado MJ (2015) Anoxic growth of Ensifer meliloti 1021 by N2O-reduction, a potential mitigation strategy. Front Microbiol 6:537

    Article  PubMed  PubMed Central  Google Scholar 

  61. Firth JR, Edwards C (1999) Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by membrane inlet mass spectrometry. J Appl Microbiol 87:353–358

    Article  CAS  PubMed  Google Scholar 

  62. Carreira C (2017) Dept Química. Universidade Nova de Lisboa, Caparica, p 264

    Google Scholar 

  63. Wunsch P, Herb M, Wieland H, Schiek UM, Zumft WG (2003) Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida. J Bacteriol 185:887–896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Borrero-de Acuna JM, Rohde M, Wissing J, Jansch L, Schobert M, Molinari G, Timmis KN, Jahn M, Jahn D (2016) Protein network of the Pseudomonas aeruginosa denitrification apparatus. J Bacteriol 198:1401–1413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

We thank Fundação para a Ciência e Tecnologia for the financial support through the project PTDC/BIA-PRO/098882/2008 (SRP) and PTDC/BBB-BQB/0129/2014 (IM), and the scholarship SFRH/BD/87898/2012 (CC). This work was supported by the Applied Molecular Biosciences Unit—UCIBIO, and Associate Laboratory for Green Chemistry—LAQV, which is financed by national funds from FCT (UIDB/04378/2020 and UIDB/50006/2020, respectively).

Author information

Authors and Affiliations

  1. Microbial Stress Lab, UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516, Caparica, Portugal

    Cíntia Carreira, Rute F. Nunes, Olga Mestre & Sofia R. Pauleta

  2. Biological Chemistry Lab, LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516, Caparica, Portugal

    Cíntia Carreira & Isabel Moura

Authors
  1. Cíntia Carreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rute F. Nunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olga Mestre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Isabel Moura
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sofia R. Pauleta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CC performed the anaerobic purifications, the spectroscopic characterization and all the activity assays. CC and RFN performed the growths in the bioreactor and nitrate/nitrite quantifications. OM performed the transcriptional analysis. IM critically read the manuscript. SRP designed and supervised the experiments and data analysis. CC and SRP wrote the manuscript and, analyzed and interpreted the data.

Corresponding author

Correspondence to Sofia R. Pauleta.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 992 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Carreira, C., Nunes, R.F., Mestre, O. et al. The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase. J Biol Inorg Chem 25, 927–940 (2020). https://doi.org/10.1007/s00775-020-01812-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-020-01812-0

Keywords

Search

Navigation