Skip to main content
SpringerLink
Log in

Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions

  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO2 content (pCO2) at the Swiss FACE (Free Air CO2 Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO2 in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO2 treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO2. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO2 treatment.

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

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. EM Baggs M Richter G Cadisch UA Hartwig (2003) ArticleTitleDenitrification in grass swards is increased under elevated atmospheric CO2. Soil Biol Biochem 35 729–732 Occurrence Handle10.1016/S0038-0717(03)00083-X Occurrence Handle1:CAS:528:DC%2BD3sXjt1GqsLY%3D

    Article  CAS  Google Scholar 

  2. B Baumann M Snozzi JB Zehnder JR van der Meer (1996) ArticleTitleDynamics of denitrification activity in Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes. J Bacteriol 178 4367–4374 Occurrence Handle1:CAS:528:DyaK28Xks1Slu74%3D Occurrence Handle8755862

    CAS  PubMed  Google Scholar 

  3. L Bedzyk T Wang RW Ye (1999) ArticleTitleThe periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification. J Bacteriol 181 2802–2806 Occurrence Handle1:CAS:528:DyaK1MXivFCjsLY%3D Occurrence Handle10217771

    CAS  PubMed  Google Scholar 

  4. BC Berks SJ Ferguson JWB Moir DJ Richardson (1995) ArticleTitleEnzymes and associated electron transport that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta 1232 97–173 Occurrence Handle1:CAS:528:DyaK2MXpvVyksrg%3D Occurrence Handle8534676

    CAS  PubMed  Google Scholar 

  5. EB Braun-Howland PA Vescio SA Nierzwicki-Bauer (1993) ArticleTitleUse of a simplified cell blot technique and 16S rRNA-directed probes for identification of common environmental isolates. Appl Environ Microbiol 159 3219–3224

    Google Scholar 

  6. M Carnol L Hogenboom M Ewajach J Remacle R Ceulemans (2002) ArticleTitleElevated atmospheric CO2 in open top chambers increases net nitrification and potential denitrification. Global Change Biol 8 590–598

    Google Scholar 

  7. JP Carter YH Hsaio S Spiro DJ Richardson (1995) ArticleTitleSoil and sediment bacteria capable of aerobic nitrate respiration. Appl Environ Microbiol 61 2852–2858 Occurrence Handle1:CAS:528:DyaK2MXnt1aitL8%3D Occurrence Handle7487017

    CAS  PubMed  Google Scholar 

  8. MA Cavigelli GP Robertson (2001) ArticleTitleRole of denitrifier diversity in rate of nitrous oxide consumption in a terrestrial ecosystem. Soil Biol Biochem 33 297–310 Occurrence Handle1:CAS:528:DC%2BD3MXhslKls7g%3D

    CAS  Google Scholar 

  9. D Chèneby L Philippot A Hartmann C Henault JC Germon (2000) ArticleTitle16S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol 34 121–128 Occurrence Handle11102689

    PubMed  Google Scholar 

  10. D Chèneby S Hallet A Mondon F Martin-Laurent JC Germon L Philippot (2003) ArticleTitleGenetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis. Microb Ecol 46 113–121 Occurrence Handle12739081

    PubMed  Google Scholar 

  11. A Clays-Josserand P Lemanceau L Philippot R Lensi (1995) ArticleTitleInfluence of two plant species (flax and tomato) on nitrogen dissimilative abilities within fluorescent Pseudomonas spp. Appl Environ Microbiol 61 1745–1749 Occurrence Handle1:CAS:528:DyaK2MXlsFyktr4%3D

    CAS  Google Scholar 

  12. A Clays-Josserand JF Ghiglione L Philippot P Lemanceau R Lensi (1999) ArticleTitleEffect of soil type and plant species on the fluorescent pseudomonads nitrate dissimilating community. Plant Soil 209 275–282 Occurrence Handle1:CAS:528:DyaK1MXlt1ems70%3D

    CAS  Google Scholar 

  13. MD Corre RR Schnabel WL Stout (2002) ArticleTitleSpatial and seasonal variation of gross nitrogen transformations and microbial biomass in a Northeastern US grassland. Soil Biol Biochem 34 445–457 Occurrence Handle1:CAS:528:DC%2BD38Xis1eiu78%3D

    CAS  Google Scholar 

  14. M Daepp D Suter JPF Almeida H Isopp UA Hartwig M Frehner H Blum J Nösberger A Lüscher (2000) ArticleTitleYield response of Lolium perenne swards to free air CO2 enrichment increased over six years in a high N input system on fertile soil. Global Change Biol 6 805–816

    Google Scholar 

  15. PR Darrah (1996) ArticleTitleRhizodeposition under ambient and elevated CO2 levels. Plant Soil 187 265–276 Occurrence Handle1:CAS:528:DyaK2sXjtlSjtrg%3D

    CAS  Google Scholar 

  16. S Delorme L Philippot V Edel-Hermann C Deulvot C Mougel P Lemanceau (2003) ArticleTitleComparative genetic diversity of the narG, nosZ, and 16S rRNA genes in fluorescent pseudomonads. Appl Environ Microbiol 69 1004–1012 Occurrence Handle1:CAS:528:DC%2BD3sXhtF2isbk%3D Occurrence Handle12571023

    CAS  PubMed  Google Scholar 

  17. CF Drury DJ McKenney WI Findlay (1991) ArticleTitleRelationships between denitrification, microbial biomass and indigenous soil properties. Soil Biol Biochem 23 751–755

    Google Scholar 

  18. DA Flanagan LG Gregory JP Carter A Karakas-Sen DJ Richardson S Spiro (1999) ArticleTitleDetection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA. FEMS Microbiol Lett 177 263–270 Occurrence Handle1:CAS:528:DyaK1MXkvVequ7Y%3D Occurrence Handle10474192

    CAS  PubMed  Google Scholar 

  19. N Fromin W Achouak JM Thiéry T Heulin (2001) ArticleTitleThe genotypic diversity of Pseudomonas brassicacearum populations isolated from roots of Arabidopsis thaliana: influence of plant genotype. FEMS Microbiol Ecol 37 21–29 Occurrence Handle1:CAS:528:DC%2BD3MXmt1Klsrs%3D

    CAS  Google Scholar 

  20. TN Gamble MR Betlach JM Tiedje (1977) ArticleTitleNumerically dominant denitrifying bacteria from world soils. Appl Environ Microbiol 33 916–939

    Google Scholar 

  21. JF Ghiglione F Gourbière P Potier L Philippot R Lensi (2000) ArticleTitleRole of respiratory nitrate reductase in ability of Pseudomonas fluorescens YT101 to colonize the rhizosphere of maize. Appl Environ Microbiol 66 4012–4016 Occurrence Handle1:CAS:528:DC%2BD3cXmsVajsLg%3D Occurrence Handle10966422

    CAS  PubMed  Google Scholar 

  22. V Gloser M Jeziková A Lüscher M Frehner H Blum J Nösberger UA Hartwig (2000) ArticleTitleSoil mineral nitrogen availability was unaffected by elevated atmospheric pCO2 in a four year old field experiment (Swiss FACE). Plant Soil 227 291–299 Occurrence Handle1:CAS:528:DC%2BD3MXovVCiug%3D%3D

    CAS  Google Scholar 

  23. LG Gregory A Karakas-Sen DJ Richardson S Spiro (2000) ArticleTitleDetection of genes for membrane-bound nitrate reductase in nitrate-respiring bacteria and in community DNA. FEMS Microbiol Lett 183 275–279 Occurrence Handle1:CAS:528:DC%2BD3cXpsFOgtw%3D%3D Occurrence Handle10675597

    CAS  PubMed  Google Scholar 

  24. LG Gregory PL Bond DJ Richardson S Spiro (2003) ArticleTitleCharacterization of nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker. Microbiology 149 229–237 Occurrence Handle1:CAS:528:DC%2BD3sXnvV2lsQ%3D%3D Occurrence Handle12576596

    CAS  PubMed  Google Scholar 

  25. J Hamelin N Fromin S Teyssier-Cuvelle S Tarnawski M Aragno (2002) ArticleTitlenifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass. Environ Microbiol 4 477–481 Occurrence Handle1:CAS:528:DC%2BD38XntFSltLc%3D Occurrence Handle12153588

    CAS  PubMed  Google Scholar 

  26. T Hebeisen V Lüscher S Zanetti BU Fischer UA Hartwig M Frehner GR Hendrey H Blum J Nösberger (1997) ArticleTitleGrowth response of Trifolium repens and Lolium perenne as monocultures and bi-species mixture to free air CO2 enrichment and management. Global Change Biol 3 149–160

    Google Scholar 

  27. L Holtan-Hartwig P Dörsch LR Bakken (2000) ArticleTitleComparison of denitrifying communities in organic soil: kinetics of NO −3 and N2O reduction. Soil Biol Biochem 32 833–843 Occurrence Handle1:CAS:528:DC%2BD3cXktFWjtrg%3D

    CAS  Google Scholar 

  28. P Ineson PA Coward UA Hartwig (1998) ArticleTitleSoil gas fluxes of N2O, CH4 and CO2 beneath Lolium perenne under elevated CO2: the Swiss free air carbon dioxide enrichment experiment. Plant Soil 198 89–95 Occurrence Handle1:CAS:528:DyaK1cXhvFKls70%3D

    CAS  Google Scholar 

  29. KH von Linne Berg H Bothe (1992) ArticleTitleThe distribution of denitrifying bacteria in soils monitored by DNA-probing. FEMS Microbiol Ecol 86 331–340

    Google Scholar 

  30. L Locatelli S Tarnawski J Hamelin P Rossi M Aragno N Fromin (2002) ArticleTitleSpecific PCR amplification for the genus Pseudomonas targeting the 3′ half of 16S rDNA and the whole 16S-23S rDNA spacer. Syst Appl Microbiol 25 220–227 Occurrence Handle1:CAS:528:DC%2BD38XosFeiu7c%3D Occurrence Handle12353876

    CAS  PubMed  Google Scholar 

  31. T Mahmood R Ali KA Malik SRA Shamsi (1997) ArticleTitleDenitrification with and without maize plants (Zea mays L) under irrigated field conditions. Biol Fertil Soil 24 323–328 Occurrence Handle1:CAS:528:DyaK2sXis1Kktr4%3D

    CAS  Google Scholar 

  32. I Mahne JM Tiedje (1995) ArticleTitleCriteria and methodology for identifying respiratory denitrifiers. Appl Environ Microbiol 61 1110–1115 Occurrence Handle1:CAS:528:DyaK2MXktV2rt70%3D

    CAS  Google Scholar 

  33. L Marilley UA Hartwig M Aragno (1999) ArticleTitleInfluence of an elevated atmospheric CO2 content on soil and rhizosphere bacterial communities beneath Lolium perenne and Trifolium repens under field conditions. Microb Ecol 38 39–49 Occurrence Handle1:CAS:528:DyaK1MXkt1OksLo%3D Occurrence Handle10384008

    CAS  PubMed  Google Scholar 

  34. P Martín-Olmedo RM Rees J Grace (2002) ArticleTitleThe influence of plants grown under elevated CO2 and N fertilisation on soil nitrogen dynamics. Global Change Biol 8 643–657

    Google Scholar 

  35. C McDevitt P Burrell LL Blackall AG McEwan (2000) ArticleTitleAerobic nitrate respiration in a nitrite oxidising bioreactor. FEMS Microbiol Ecol 184 113–118 Occurrence Handle1:CAS:528:DC%2BD3cXht1Oju7g%3D

    CAS  Google Scholar 

  36. C Moreno-Vivián SJ Ferguson (1998) ArticleTitleDefinition and distinction between assimilatory, dissimilatory and respiratory pathways. Mol Microbiol 29 664–666 Occurrence Handle9720883

    PubMed  Google Scholar 

  37. JW Nijburg HJ Laanbroek (1997) ArticleTitleThe influence of Glyceria maxima and nitrate input on the composition and nitrate metabolism of the dissimilatory nitrate-reducing bacterial community. FEMS Microbiol Ecol 22 57–63 Occurrence Handle1:CAS:528:DyaK2sXktl2nug%3D%3D

    CAS  Google Scholar 

  38. JW Nijburg MJL Coolen S Gerards PJAK Gunnewiek HJ Laanbroek (1997) ArticleTitleEffects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community. Appl Environ Microbiol 63 931–937 Occurrence Handle1:CAS:528:DyaK2sXhsV2ls7o%3D

    CAS  Google Scholar 

  39. B Nogales KN Timmis DB Nedwell AM Osborn (2002) ArticleTitleDetection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription–PCR amplification from mRNA. Appl Environ Microbiol 68 5017–5025 Occurrence Handle1:CAS:528:DC%2BD38XnvFClu74%3D Occurrence Handle12324352

    CAS  PubMed  Google Scholar 

  40. S Parry P Renault C Chenu R Lensi (1999) ArticleTitleDenitrification in pasture and cropped soil clods as affected by spore space structure. Soil Biol Biochem 31 493–501 Occurrence Handle1:CAS:528:DyaK1MXitlaitbk%3D

    CAS  Google Scholar 

  41. L Philippot (2002) ArticleTitleDenitrifying genes in bacterial and archeal genomes. Biochim Biophys Acta 1577 355–376 Occurrence Handle1:CAS:528:DC%2BD38XnsVSmt78%3D Occurrence Handle12359326

    CAS  PubMed  Google Scholar 

  42. L Philippot O Højberg (1999) ArticleTitleDissimilatory nitrate reductases in bacteria. Biochim Biophys Acta 1446 1–23 Occurrence Handle1:CAS:528:DyaK1MXltVyrtrw%3D Occurrence Handle10395915

    CAS  PubMed  Google Scholar 

  43. L Philippot P Mirleau S Mazurier S Siblot A Hartmann P Lemanceau JC Germon (2001) ArticleTitleCharacterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes. Biochim Biophys Acta 1517 436–440 Occurrence Handle1:CAS:528:DC%2BD3MXhtFentb0%3D Occurrence Handle11342223

    CAS  PubMed  Google Scholar 

  44. L Philippot S Piutti F Martin-Laurent S Hallet JC Germon (2002) ArticleTitleMolecular analysis of the nitrate-reducing community from unplanted and maize-planted soil. Appl Environ Microbiol 68 6121–6128 Occurrence Handle1:CAS:528:DC%2BD38XptlartL4%3D Occurrence Handle12450836

    CAS  PubMed  Google Scholar 

  45. L Potter H Angove D Richardson J Cole (2001) ArticleTitleNitrate reduction in the periplasm of gram-negative bacteria. Adv Microb Physiol 45 51–112 Occurrence Handle1:CAS:528:DC%2BD3MXls1eltbk%3D Occurrence Handle11450112

    CAS  PubMed  Google Scholar 

  46. K Prade G Trolldenier (1990) ArticleTitleDenitrification in the rhizosphere of rice and wheat seedlings as influenced by plant K status, air-filled porosity and substrate organic matter. Soil Biol Biochem 22 76–773

    Google Scholar 

  47. DJ Richardson BC Berks DA Russel S Spiro CJ Taylor (2001) ArticleTitleFunctional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58 165–178 Occurrence Handle1:CAS:528:DC%2BD3MXivFOltrw%3D Occurrence Handle11289299

    CAS  PubMed  Google Scholar 

  48. M Richter UA Hartwig E Frossard J Nösberger G Cadisch (2003) ArticleTitleGross fluxes of nitrogen in grassland soil exposed to elevated atmospheric pCO2 for seven years. Soil Biol Biochem 35 1325–1335 Occurrence Handle1:CAS:528:DC%2BD3sXntVGntbo%3D

    CAS  Google Scholar 

  49. DR Smart K Ritchie JM Stark B Bugbee (1997) ArticleTitleEvidence that elevated CO2 levels can indirectly increase rhizosphere denitrifier activity. Appl Environ Microbiol 63 4621–4624 Occurrence Handle1:CAS:528:DyaK2sXnt12nu7g%3D Occurrence Handle11536820

    CAS  PubMed  Google Scholar 

  50. V Stewart Y Lu AJ Darwin (2002) ArticleTitlePeriplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12. J Bacteriol 184 1314–1323 Occurrence Handle1:CAS:528:DC%2BD38XhtlKntr8%3D Occurrence Handle11844760

    CAS  PubMed  Google Scholar 

  51. DT Strong IRP Fillery (2002) ArticleTitleDenitrification response to nitrate concentrations in sandy soils. Soil Biol Biochem 34 945–954 Occurrence Handle1:CAS:528:DC%2BD38XksFKntLc%3D

    CAS  Google Scholar 

  52. S Tarnawski J Hamelin L Locatelli M Aragno N Fromin (2003) ArticleTitleExamination of Gould’s modified S1 (mS1) selective medium and Angle’s non-selective medium for collecting diversity of Pseudomonas spp. in soil and root environments. FEMS Microbiol Ecol 45 97–104 Occurrence Handle1:CAS:528:DC%2BD3sXlsVaju74%3D

    CAS  Google Scholar 

  53. JM Tiedje (1988) Ecology of denitrification and dissimilatory nitrate reduction to ammonium. AJB Zehnder (Eds) Biology of Anaerobic Bacteria John Wiley and Sons New York 179–244

    Google Scholar 

  54. M van Oorschot N van Gaalen E Maltby N Mockler A Spink JTA Verhoeven (2000) ArticleTitleExperimental manipulation of water levels in two French riverine grassland soils. Acta Oecol Int J Ecol 21 49–62

    Google Scholar 

  55. BR Vázquez de Aldana F Berendse (1997) ArticleTitleNitrogen-use efficiency in six perennial grasses from contrasting habitats. Funct Ecol 11 619–626

    Google Scholar 

  56. PM Vitousek HA Mooney J Lubchenco JM Melillo (1997) ArticleTitleHuman domination of Earth’s ecosystem. Science 277 494–499 Occurrence Handle1:CAS:528:DyaK2sXkvVektLs%3D

    CAS  Google Scholar 

  57. DR Zak SKS Pregitzer JS King WE Holmes (2000) ArticleTitleElevated atmospheric CO2, fine roots and the response of soil microorganisms: a review and hypothesis. New Phytol 147 201–222 Occurrence Handle1:CAS:528:DC%2BD3cXms1yltLs%3D

    CAS  Google Scholar 

  58. S Zanetti UA Hartwig C van Kessel A Lüscher T Hebeisen M Frehner BU Fisher GR Hendrey H Blum J Nösberger (1997) ArticleTitleDoes nitrogen nutrition restrict the CO2 response of fertile grassland lacking legumes? Oecologia 112 17–25

    Google Scholar 

  59. WG Zumft (1997) ArticleTitleCell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61 533–616 Occurrence Handle1:CAS:528:DyaK2sXotVymtr8%3D Occurrence Handle9409151

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Swiss National Science Foundation (grant numbers 3100-055899.98 and 31-68208.02). We are also grateful to the Swiss National Centre of Competence in Research (NCCR) “Plant Survival.” We thank Marie-Laure Heusler for technical assistance, Jakob Zopfi and Raymond Flynn for English corrections, and Jacqueline Moret for statistical analysis.

Author information

Authors and Affiliations

  1. Laboratoire de Microbiologie, Université de Neuchâtel, rue Emile Argand 11, 2007, Neuchâtel, Switzerland

    L. Roussel-Delif, S. Tarnawski, J. Hamelin, M. Aragno & N. Fromin

  2. UMRA111, Microbiologie des Sols-Géosols, Laboratoire de Microbiologie des Sols, Institut National de la Recherche Agronomique, 21055, Dijon Cedex, France

    L. Philippot

Authors
  1. L. Roussel-Delif
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Tarnawski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Hamelin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. Philippot
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Aragno
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. N. Fromin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to N. Fromin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Roussel-Delif, L., Tarnawski, S., Hamelin, J. et al. Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions. Microb Ecol 49, 63–72 (2005). https://doi.org/10.1007/s00248-003-0228-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-003-0228-3

Keywords

Search

Navigation