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Applied and Environmental Microbiology, August 1998, p. 2958-2965, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of beta -Subgroup Proteobacterial Ammonia Oxidizer Populations in Soil by Denaturing Gradient Gel Electrophoresis Analysis and Hierarchical Phylogenetic Probing

John R. Stephen,1,2,3 George A. Kowalchuk,3 Mary-Ann V. Bruns,1,4,dagger Allison E. McCaig,1 Carol J. Phillips,1 T. Martin Embley,2 and James I. Prosser1,*

Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland AB25 2ZD,1 and Department of Zoology, Natural History Museum, London SW7 5BD,2 United Kingdom; Department of Plant Microorganism Interactions, Netherlands Institute of Ecology, 6666 ZG Heteren, The Netherlands3; and Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824-13254

Received 4 March 1998/Accepted 11 May 1998

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta -subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all beta -subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta -subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom. Phone: 44 1224 273148. Fax: 44 1224 273144. E-mail: j.prosser{at}ac.uk.aberdeen.

dagger Present address: Department of Land, Air and Water Resources, University of California, Davis, CA 95616.

Applied and Environmental Microbiology, August 1998, p. 2958-2965, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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