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Microbial Ecology

, Volume 49, Issue 1, pp 63–72 | Cite as

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

  • L. Roussel-Delif
  • S. Tarnawski
  • J. Hamelin
  • L. Philippot
  • M. Aragno
  • N. FrominEmail author
Article

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.

Keywords

Denitrification Nitrate Reductase Perennial Grass Nitrate Reducer napA 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

References

  1. 1.
    Baggs, EM, Richter, M, Cadisch, G, Hartwig, UA 2003Denitrification in grass swards is increased under elevated atmospheric CO2.Soil Biol Biochem35729732CrossRefGoogle Scholar
  2. 2.
    Baumann, B, Snozzi, M, Zehnder, JB, van der Meer, JR 1996Dynamics of denitrification activity in Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.J Bacteriol17843674374PubMedGoogle Scholar
  3. 3.
    Bedzyk, L, Wang, T, Ye, RW 1999The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification.J Bacteriol18128022806PubMedGoogle Scholar
  4. 4.
    Berks, BC, Ferguson, SJ, Moir, JWB, Richardson, DJ 1995Enzymes and associated electron transport that catalyse the respiratory reduction of nitrogen oxides and oxyanions.Biochim Biophys Acta123297173PubMedGoogle Scholar
  5. 5.
    Braun-Howland, EB, Vescio, PA, Nierzwicki-Bauer, SA 1993Use of a simplified cell blot technique and 16S rRNA-directed probes for identification of common environmental isolates.Appl Environ Microbiol15932193224Google Scholar
  6. 6.
    Carnol, M, Hogenboom, L, Ewajach, M, Remacle, J, Ceulemans, R 2002Elevated atmospheric CO2 in open top chambers increases net nitrification and potential denitrification.Global Change Biol8590598Google Scholar
  7. 7.
    Carter, JP, Hsaio, YH, Spiro, S, Richardson, DJ 1995Soil and sediment bacteria capable of aerobic nitrate respiration.Appl Environ Microbiol6128522858PubMedGoogle Scholar
  8. 8.
    Cavigelli, MA, Robertson, GP 2001Role of denitrifier diversity in rate of nitrous oxide consumption in a terrestrial ecosystem.Soil Biol Biochem33297310Google Scholar
  9. 9.
    Chèneby, D, Philippot, L, Hartmann, A, Henault, C, Germon, JC 200016S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils.FEMS Microbiol Ecol34121128PubMedGoogle Scholar
  10. 10.
    Chèneby, D, Hallet, S, Mondon, A, Martin-Laurent, F, Germon, JC, Philippot, L 2003Genetic characterization of the nitrate reducing community based on narG nucleotide sequence analysis.Microb Ecol46113121PubMedGoogle Scholar
  11. 11.
    Clays-Josserand, A, Lemanceau, P, Philippot, L, Lensi, R 1995Influence of two plant species (flax and tomato) on nitrogen dissimilative abilities within fluorescent Pseudomonas spp.Appl Environ Microbiol6117451749Google Scholar
  12. 12.
    Clays-Josserand, A, Ghiglione, JF, Philippot, L, Lemanceau, P, Lensi, R 1999Effect of soil type and plant species on the fluorescent pseudomonads nitrate dissimilating community.Plant Soil209275282Google Scholar
  13. 13.
    Corre, MD, Schnabel, RR, Stout, WL 2002Spatial and seasonal variation of gross nitrogen transformations and microbial biomass in a Northeastern US grassland.Soil Biol Biochem34445457Google Scholar
  14. 14.
    Daepp, M, Suter, D, Almeida, JPF, Isopp, H, Hartwig, UA, Frehner, M, Blum, H, Nösberger, J, Lüscher, A 2000Yield 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 Biol6805816Google Scholar
  15. 15.
    Darrah, PR 1996Rhizodeposition under ambient and elevated CO2 levels.Plant Soil187265276Google Scholar
  16. 16.
    Delorme, S, Philippot, L, Edel-Hermann, V, Deulvot, C, Mougel, C, Lemanceau, P 2003Comparative genetic diversity of the narG, nosZ, and 16S rRNA genes in fluorescent pseudomonads.Appl Environ Microbiol6910041012PubMedGoogle Scholar
  17. 17.
    Drury, CF, McKenney, DJ, Findlay, WI 1991Relationships between denitrification, microbial biomass and indigenous soil properties.Soil Biol Biochem23751755Google Scholar
  18. 18.
    Flanagan, DA, Gregory, LG, Carter, JP, Karakas-Sen, A, Richardson, DJ, Spiro, S 1999Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA.FEMS Microbiol Lett177263270PubMedGoogle Scholar
  19. 19.
    Fromin, N, Achouak, W, Thiéry, JM, Heulin, T 2001The genotypic diversity of Pseudomonas brassicacearum populations isolated from roots of Arabidopsis thaliana: influence of plant genotype.FEMS Microbiol Ecol372129Google Scholar
  20. 20.
    Gamble, TN, Betlach, MR, Tiedje, JM 1977Numerically dominant denitrifying bacteria from world soils.Appl Environ Microbiol33916939Google Scholar
  21. 21.
    Ghiglione, JF, Gourbière, F, Potier, P, Philippot, L, Lensi, R 2000Role of respiratory nitrate reductase in ability of Pseudomonas fluorescens YT101 to colonize the rhizosphere of maize.Appl Environ Microbiol6640124016PubMedGoogle Scholar
  22. 22.
    Gloser, V, Jeziková, M, Lüscher, A, Frehner, M, Blum, H, Nösberger, J, Hartwig, UA 2000Soil mineral nitrogen availability was unaffected by elevated atmospheric pCO2 in a four year old field experiment (Swiss FACE).Plant Soil227291299Google Scholar
  23. 23.
    Gregory, LG, Karakas-Sen, A, Richardson, DJ, Spiro, S 2000Detection of genes for membrane-bound nitrate reductase in nitrate-respiring bacteria and in community DNA.FEMS Microbiol Lett183275279PubMedGoogle Scholar
  24. 24.
    Gregory, LG, Bond, PL, Richardson, DJ, Spiro, S 2003Characterization of nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker.Microbiology149229237PubMedGoogle Scholar
  25. 25.
    Hamelin, J, Fromin, N, Teyssier-Cuvelle, S, Tarnawski, S, Aragno, M 2002nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass.Environ Microbiol4477481PubMedGoogle Scholar
  26. 26.
    Hebeisen, T, Lüscher, V, Zanetti, S, Fischer, BU, Hartwig, UA, Frehner, M, Hendrey, GR, Blum, H, Nösberger, J 1997Growth response of Trifolium repens and Lolium perenne as monocultures and bi-species mixture to free air CO2 enrichment and management.Global Change Biol3149160Google Scholar
  27. 27.
    Holtan-Hartwig, L, Dörsch, P, Bakken, LR 2000Comparison of denitrifying communities in organic soil: kinetics of NO 3 and N2O reduction.Soil Biol Biochem32833843Google Scholar
  28. 28.
    Ineson, P, Coward, PA, Hartwig, UA 1998Soil gas fluxes of N2O, CH4 and CO2 beneath Lolium perenne under elevated CO2: the Swiss free air carbon dioxide enrichment experiment.Plant Soil1988995Google Scholar
  29. 29.
    von Linne Berg, KH, Bothe, H 1992The distribution of denitrifying bacteria in soils monitored by DNA-probing.FEMS Microbiol Ecol86331340Google Scholar
  30. 30.
    Locatelli, L, Tarnawski, S, Hamelin, J, Rossi, P, Aragno, M, Fromin, N 2002Specific PCR amplification for the genus Pseudomonas targeting the 3′ half of 16S rDNA and the whole 16S-23S rDNA spacer.Syst Appl Microbiol25220227PubMedGoogle Scholar
  31. 31.
    Mahmood, T, Ali, R, Malik, KA, Shamsi, SRA 1997Denitrification with and without maize plants (Zea mays L) under irrigated field conditions.Biol Fertil Soil24323328Google Scholar
  32. 32.
    Mahne, I, Tiedje, JM 1995Criteria and methodology for identifying respiratory denitrifiers.Appl Environ Microbiol6111101115Google Scholar
  33. 33.
    Marilley, L, Hartwig, UA, Aragno, M 1999Influence of an elevated atmospheric CO2 content on soil and rhizosphere bacterial communities beneath Lolium perenne and Trifolium repens under field conditions.Microb Ecol383949PubMedGoogle Scholar
  34. 34.
    Martín-Olmedo, P, Rees, RM, Grace, J 2002The influence of plants grown under elevated CO2 and N fertilisation on soil nitrogen dynamics.Global Change Biol8643657Google Scholar
  35. 35.
    McDevitt, C, Burrell, P, Blackall, LL, McEwan, AG 2000Aerobic nitrate respiration in a nitrite oxidising bioreactor.FEMS Microbiol Ecol184113118Google Scholar
  36. 36.
    Moreno-Vivián, C, Ferguson, SJ 1998Definition and distinction between assimilatory, dissimilatory and respiratory pathways.Mol Microbiol29664666PubMedGoogle Scholar
  37. 37.
    Nijburg, JW, Laanbroek, HJ 1997The influence of Glyceria maxima and nitrate input on the composition and nitrate metabolism of the dissimilatory nitrate-reducing bacterial community.FEMS Microbiol Ecol225763Google Scholar
  38. 38.
    Nijburg, JW, Coolen, MJL, Gerards, S, Gunnewiek, PJAK, Laanbroek, HJ 1997Effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community.Appl Environ Microbiol63931937Google Scholar
  39. 39.
    Nogales, B, Timmis, KN, Nedwell, DB, Osborn, AM 2002Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription–PCR amplification from mRNA.Appl Environ Microbiol6850175025PubMedGoogle Scholar
  40. 40.
    Parry, S, Renault, P, Chenu, C, Lensi, R 1999Denitrification in pasture and cropped soil clods as affected by spore space structure.Soil Biol Biochem31493501Google Scholar
  41. 41.
    Philippot, L 2002Denitrifying genes in bacterial and archeal genomes.Biochim Biophys Acta1577355376PubMedGoogle Scholar
  42. 42.
    Philippot, L, Højberg, O 1999Dissimilatory nitrate reductases in bacteria.Biochim Biophys Acta1446123PubMedGoogle Scholar
  43. 43.
    Philippot, L, Mirleau, P, Mazurier, S, Siblot, S, Hartmann, A, Lemanceau, P, Germon, JC 2001Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes.Biochim Biophys Acta1517436440PubMedGoogle Scholar
  44. 44.
    Philippot, L, Piutti, S, Martin-Laurent, F, Hallet, S, Germon, JC 2002Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soil.Appl Environ Microbiol6861216128PubMedGoogle Scholar
  45. 45.
    Potter, L, Angove, H, Richardson, D, Cole, J 2001Nitrate reduction in the periplasm of gram-negative bacteria.Adv Microb Physiol4551112PubMedGoogle Scholar
  46. 46.
    Prade, K, Trolldenier, G 1990Denitrification in the rhizosphere of rice and wheat seedlings as influenced by plant K status, air-filled porosity and substrate organic matter.Soil Biol Biochem2276773Google Scholar
  47. 47.
    Richardson, DJ, Berks, BC, Russel, DA, Spiro, S, Taylor, CJ 2001Functional, biochemical and genetic diversity of prokaryotic nitrate reductases.Cell Mol Life Sci58165178PubMedGoogle Scholar
  48. 48.
    Richter, M, Hartwig, UA, Frossard, E, Nösberger, J, Cadisch, G 2003Gross fluxes of nitrogen in grassland soil exposed to elevated atmospheric pCO2 for seven years.Soil Biol Biochem3513251335Google Scholar
  49. 49.
    Smart, DR, Ritchie, K, Stark, JM, Bugbee, B 1997Evidence that elevated CO2 levels can indirectly increase rhizosphere denitrifier activity.Appl Environ Microbiol6346214624PubMedGoogle Scholar
  50. 50.
    Stewart, V, Lu, Y, Darwin, AJ 2002Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12.J Bacteriol18413141323PubMedGoogle Scholar
  51. 51.
    Strong, DT, Fillery, IRP 2002Denitrification response to nitrate concentrations in sandy soils.Soil Biol Biochem34945954Google Scholar
  52. 52.
    Tarnawski, S, Hamelin, J, Locatelli, L, Aragno, M, Fromin, N 2003Examination 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 Ecol4597104Google Scholar
  53. 53.
    Tiedje, JM 1988Ecology of denitrification and dissimilatory nitrate reduction to ammonium.Zehnder, AJB eds. Biology of Anaerobic BacteriaJohn Wiley and SonsNew York179244Google Scholar
  54. 54.
    van Oorschot, M, van Gaalen, N, Maltby, E, Mockler, N, Spink, A, Verhoeven, JTA 2000Experimental manipulation of water levels in two French riverine grassland soils.Acta Oecol Int J Ecol214962Google Scholar
  55. 55.
    Vázquez de Aldana, BR, Berendse, F 1997Nitrogen-use efficiency in six perennial grasses from contrasting habitats.Funct Ecol11619626Google Scholar
  56. 56.
    Vitousek, PM, Mooney, HA, Lubchenco, J, Melillo, JM 1997Human domination of Earth’s ecosystem.Science277494499Google Scholar
  57. 57.
    Zak, DR, Pregitzer, SKS, King, JS, Holmes, WE 2000Elevated atmospheric CO2, fine roots and the response of soil microorganisms: a review and hypothesis.New Phytol147201222Google Scholar
  58. 58.
    Zanetti, S, Hartwig, UA, van Kessel, C, Lüscher, A, Hebeisen, T, Frehner, M, Fisher, BU, Hendrey, GR, Blum, H, Nösberger, J 1997Does nitrogen nutrition restrict the CO2 response of fertile grassland lacking legumes?Oecologia1121725Google Scholar
  59. 59.
    Zumft, WG 1997Cell biology and molecular basis of denitrification.Microbiol Mol Biol Rev61533616PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • L. Roussel-Delif
    • 1
  • S. Tarnawski
    • 1
  • J. Hamelin
    • 1
  • L. Philippot
    • 2
  • M. Aragno
    • 1
  • N. Fromin
    • 1
    Email author
  1. 1.Laboratoire de MicrobiologieUniversité de NeuchâtelNeuchâtelSwitzerland
  2. 2.UMRA111, Microbiologie des Sols-Géosols, Laboratoire de Microbiologie des SolsInstitut National de la Recherche AgronomiqueDijon CedexFrance

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