ANALYSIS OF BETA-SUBGROUP PROTEOBACTERIAL AMMONIA OXIDIZER POPULATIONS IN SOIL BY DENATURING GRADIENT GEL-ELECTROPHORESIS ANALYSIS AND HIERARCHICAL PHYLOGENETIC PROBING

A combination of denaturing gradient gel electrophoresis (DGGE) and ol igonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgro mp proteobacterial ammonia oxidizers. PCR primers specific to this gro up were used to amplify 16S ribosomal DIVA (rDNA) from soils maintaine d 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 spe cific 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. Presser , and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and clo sely related nontarget sequences indicated a requirement for high-stri ngency hybridization conditions to distinguish between different clust ers, DGGE banding patterns suggested the presence of Nitrosomonas clus ter 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but re sults mere ambiguous because of overlapping banding patterns, Unambigu ous hand identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled prob es, The relative intensities of hybridization signals provided informa tion on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira clu ster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitro sospira 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 agre ement with a previous molecular study (J, R Stephen, A. E. McCaig, Z. Smith, J. I, Presser, and T, M. Embley, Appl, Environ. Microbiol 62:41 47-4154, 1996) of the same sites, which demonstrated the presence of t he same four clusters of ammonia oxidizers and indicated that selectio n might be occurring for clusters 2 and 3 at acid and neutral pHs, res pectively. The two studies used different sets of PCR primers for ampl ification of 16S rDNA sequences from soil, and the similar findings su ggest that PCR bias was unlikely to be a significant factor, The prese nt 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 Nitrosos pira populations, and suggests that Nitrosospira cluster 2 may be of s ignificance for ammonia-oxidizing activity in acid soils.