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

Abstract

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 β-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 β-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 ofNitrosomonas cluster 6a and Nitrosospiraclusters 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 theNitrosospira 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 Nitrosomonascluster 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 β-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.

Quantitative analyses of the abundance and composition of ammonia‐oxidizing bacteria and ammonia‐oxidizing archaea of a Chinese upland red soil under long‐term fertilization practices

“…Phylogenetic analysis showed that Nitrosospira cluster 3 dominated in soils, which is in agreement with previous study that Nitrosospira cluster 3 sequences were most common in agricultural fields (Kowalchuk et al, 2000). However, our result was inconsistent reports of selection of Nitrosospira cluster 2 in acidic soils (Stephen et al, 1996;1998;Kowalchuk et al, 2000). Given this discrepancy, additional sequence analysis was carried out for low pH treatments but no Nitrosospira cluster 2 sequence was found.…”

Growth, activity and temperature responses of ammonia‐oxidizing archaea and bacteria in soil microcosms

“…In previous studies of this soil (e.g. Stephen et al, 1998), all sequences amplified by the bacterial ammonia oxidizer primers used here fell within recognized bacterial ammonia oxidizer phylogenetic groups. However, to determine identity of crenarchaeal DGGE bands, clone sequences (CBS16S1-6) representing the six dominant band positions in 16S rRNA gene DGGE profiles ( Fig.…”

Diversity and composition of tropical soil nitrifiers across a plant diversity gradient and among land‐use types

“…However, it is important to note that even in the intensively managed system utilized by Bruns et al (1999), changes in qualitative phylogenetic diversity between native and cultivated soils are not always evident (Phillips et al 2000). Other studies suggest that AOB may be sensitive to the type of fertilizer application (Oved et al 2001), soil pH (Stephen et al 1998;Kowalchuk et al 2000), soil temperature (Avrahami et al 2003), and the heterogeneity of soil characteristics (Webster et al 2002), all of which can change differentially depending on management.…”