Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in native, tilled, and successional soils

Abstract

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.

FIG. 1

Neighbor-joining tree (22) showing relationships of partial 16S rDNA sequences from soil communities with reference AAO sequences and those of other selected β-proteobacteria. The scale equals 10% estimated substitutions calculated by the Kimura correction (9). Bootstrap values represent percentages from 100 parsimony–maximum-likelihood analyses (29). Cloned sequences from native and tilled soils are designated KZOO_D and KZOO_H, respectively. Abbreviations representing other AAO sequences are as previously described (27).

FIG. 2

DGGE band patterns of 16S rDNA PCR amplification products obtained with the CTO_PCR primers (10) and community DNA. DGGE gels were stained with ethidium bromide and photographed under UV illumination. Lanes: 1, 3, 5, and 7, PCR products from 1994 samples; 2, 4, 6, and 8, PCR products from 1995 samples; 1 and 2, tilled, replicate plot 5; 3 and 4, tilled, replicate plot 6; 5 and 6, native (never-tilled), replicate plot 3; 7 and 8, native (never-tilled), replicate plot 4; 9 through 12, PCR products from 1995 samples; 9, fertilized successional microplot within replicate plot 1; 10, unfertilized successional, replicate plot 1; 11, fertilized successional microplot within replicate plot 3; 12, unfertilized successional, replicate plot 3. The arrows on the right indicate the locations of unique bands in lane 12.

FIG. 3

Autoradiogram of a DGGE blot hybridized with a ³²P-labeled Nsp436 probe specific for all Nitrosospira sequences (Table 1). The blot contains DNA from the gel shown in Fig. 2, and the lane designations are the same as in Fig. 2.

FIG. 4

Autoradiogram of a DGGE blot hybridized with a ³²P-labeled NspCL3_454 probe for Nitrosospira cluster 3 (Table 1). The blot contains DNA from the gel shown in Fig. 2, and the lane designations are the same as in Fig. 2.