MOLECULAR ANALYSIS OF ENRICHMENT CULTURES OF MARINE AMMONIA OXIDIZERS

Marine ammonia oxidising bacteria were enriched by incubation of sea w ater, amended with ammonium sulphate, and subsequent subculture in liq uid inorganic medium. PCR primers were designed to be specific for rDN A sequences from ammonia oxidisers belonging to the beta-sub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PC R products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the pr esence of three new lineages within the clade defined by sequences of cultured beta-sub-group ammonia oxidisers. Two of the new lineages sho wed moderate similarity to sequences from pure cultures of ammonia oxi disers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira, which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the beta-proteobacteria ammonia oxidisers. The resu lts also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolatio n of pure cultures, significantly reducing the time required and facil itating quantitative assessment of relatedness of strains.