16S ribosomal DNA analysis of marine ammonia-oxidising bacteria
Nitrification is central to the global cycling of nitrogen and is important for primary production and other processes in marine systems, where nitrogen is generally the limiting nutrient. The majority of studies on nitrification, however, have focused on terrestrial systems, where this process can cause significant losses of N-based fertilisers from agricultural land. Comparatively little is known of nitrification in marine systems and the organisms involved. In addition, technical difficulties in isolation of pure cultures of ammonia-oxidising bacteria have severely restricted studies of species diversity and community structure of these organisms. In this study, molecular technology, based on the 16S ribosomal RNA (rRNA) molecule was applied to the characterisation of marine communities of ammonia-oxidising bacteria. PCR primers were designed for specific amplification of the rRNA gene (rDNA) from ammonia-oxidisers belonging to the beta-subdivision of the Proteobacteria. These primers were used to characterise both enrichment cultures and communities within polluted and unpolluted sediment samples. These studies indicated considerable diversity of beta-subgroup ammonia-oxidisers within marine systems which has not previously been detected. It was also shown that enrichment and isolation techniques select for strains belonging to the genus Nitrosomonas while the majority of sequences obtained by direct analysis of rDNA amplified from total genomic extracts belonged to the genus Nitrosospira. In addition, novel isolation methods were developed which considerably reduced the level of heterotrophic contamination and greatly facilitated isolation of pure cultures. In situ probing, using fluorescently labelled rRNA oligonucleotide probes, indicated that CCD microscopy is less sensitive than UV microscopy alone due to quenching of the signal.