ANALYSIS OF AMMONIA-OXIDIZING BACTERIA OF THE BETA-SUBDIVISION OF THECLASS PROTEOBACTERIA IN COASTAL SAND DUNES BY DENATURING GRADIENT GEL-ELECTROPHORESIS AND SEQUENCING OF PCR-AMPLIFIED 16S RIBOSOMAL DNA FRAGMENTS

Denaturing gradient gel electrophoresis (DGGE) is a powerful and conve nient tool for analyzing the sequence diversity of complex natural mic robial populations, DGGE was evaluated for the identification of ammon ia oxidizers of the beta subdivision of the Proteobacteria based on th e mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR o f DNA extracted from coastal sand dunes, Degenerate PCR primers, CTO18 9f-GC and CTO654r, incorporating a 5' GC clamp, were designed to ampli fy a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains, The primers were tested against a representative selection of clones and cultures encompassing the currently recognized P-subdivision ammon ia oxidizer 16S rDNA sequence diversity, Analysis of these products by DGGE revealed that while many of the sequences could be separated, so me which were known to be different migrated similarly in the denatura nt system used, The CTO primer pair was used to amplify 16S rDNA seque nces from DNA extracted from soil sampled from Dutch coastal dune loca tions differing in pH and distance from the beach, The derived DGGE pa tterns were reproducible across multiple DNA isolations and PCRs, Ammo nia oxidizer-like sequences from different phylogenetic groupings isol ated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples, Bands from the DGGE gels of environmental samples were excised, reamplified, and dire ctly sequenced, revealing strong similarity or identity of the recover ed products to the corresponding regions of library clones, Six of the seven recognized sequence clusters of beta-subdivision ammonia oxidiz ers were detected in the dune systems, and differences in community st ructure between some sample sites were demonstrated, The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only sit e where sequences related to the Nitrosomonas genus could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils, Such differences in community structure may r eflect physiological differences within beta-subdivision ammonia oxidi zers, with consequent effects on nitrification rates in response to ke y environmental factors.