Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments

Abstract

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the ␤ subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5 GC clamp, were designed to amplify 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 ␤-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated 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 directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven recognized sequence clusters of ␤-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure 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 site 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 reflect physiological differences within ␤-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef

“…This increases the complexity of DGGE profiles and makes interpretation difficult and may provide another reason for the heterogeneity of the DGGE patterns resulting in the separation of the samples in nMDS analysis. Double bands may be related to degenerate positions in the primers (Kowalchuk et al, 1997) or alternatively they can be caused by abortion of the elongation reaction during PCR caused by the GC-clamp forming stable dimers and hairpins (Nübel et al, 1996;Muyzer et al, 1998).…”

The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria

“…For DGGE analysis of AOB communities, 16S rRNA genes were amplified from extracted DNA using a nested PCR approach (Freitag et al, 2006). Primary amplification used CTO189f and CTO654r PCR primers (Kowalchuk et al, 1997) and amplicons were nested with P3 (357f-GC) and P2 (518r) (Muyzer et al, 1993). Primary amplification of archaeal 16S rRNA genes was performed using primers A109f (Großkopf et al, 1998) and a modified version of 1492r (Lane, 1991) to avoid mismatches with soil crenarchaeal sequences over this region (G.W.…”

Effects of management regime and plant species on the enzyme activity and genetic structure of N‐fixing, denitrifying and nitrifying bacterial communities in grassland soils

“…The structure of the ammonia oxidizing community was analysed by PCR-DGGE. Amplification of 16S rRNA gene fragments from extracted soil DNA was achieved by a primary amplification with CTO189f and CTO654r primers (Kowalchuk et al, 1997) that are specific for the majority of betaproteobacterial ammonia oxidizers, and with a secondary nested amplification using eubacterial 357f-GC and 518r primers (Muyzer et al, 1993). CTO and eubacterial primers amplified 465 bp and 161 bp fragments respectively.…”