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Applied and Environmental Microbiology, July 1999, p. 2994-3000, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils

Mary Ann Bruns,1,* John R. Stephen,2,3,dagger George A. Kowalchuk,3 James I. Prosser,2 and Eldor A. Paul1

National Science Foundation Center for Microbial Ecology and Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 488241; Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland2; and Netherlands Institute of Ecology, 6666 ZG Heteren, The Netherlands3

Received 22 December 1998/Accepted 25 April 1999

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospira cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.

* Corresponding author. Present address: Department of Land, Air and Water Resources, University of California, Davis, CA 95616-8627. Phone: (530) 752-0146. Fax: (530) 752-1552. E-mail: mvbruns{at}ucdavis.edu.

dagger Present address: Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37932-2575.

Applied and Environmental Microbiology, July 1999, p. 2994-3000, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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