Abstract
Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r was the most suitable primer set for multiple molecular assessments of anammox bacteria in freshwater environments.
Highlights
Even before the discovery of anaerobic ammonia oxidizing bacteria as Planctomycetes [1] and elucidation of their metabolic pathways, physiology and morphology [2,3], the anammox process was recognized by nutrient profiles and thermodynamic calculations [4,5,6]
The results demonstrated that in all cases, patterns generated by bacteria-specific primers (341f-GC/518r) were distinct from those generated by the nested anammox PCR protocol for denaturing gradient gel electrophoresis (DGGE) (Fig. 1)
Of the 20 bands selected for sequencing, all three analyzed bands from SedBr, one band from SedBl and three bands from GW were affiliated with anammox bacteria (Fig. 1), demonstrating that this nested PCR design resulted in the enrichment of anammox bacterial amplicons
Summary
Even before the discovery of anaerobic ammonia oxidizing (anammox) bacteria as Planctomycetes [1] and elucidation of their metabolic pathways, physiology and morphology [2,3], the anammox process was recognized by nutrient profiles and thermodynamic calculations [4,5,6]. Many reports have demonstrated the widespread occurrence and contribution of anammox bacteria in a variety of natural ecosystems [8,9,10,11,12]. Anammox bacteria are important in the global nitrogen cycle, and responsible for high nitrogen losses: ,50% in marine sediments [13,14,15,16], ,40% in contaminated groundwater [17] and 4–37% in terrestrial habitats [18]. These findings demonstrate the important role of anammox bacteria in natural environments
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