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DenitrifyingEpsilonproteobacteria may dominate nitrogen loss processes in marine habitats with intense redox gradients, but assessment of their importance is limited by the currently available primers for nitrite reductase genes. Nine new primers targeting thenirS gene of denitrifyingEpsilonproteobacteria were designed and tested for use in sequencing and quantitative PCR on two microbial mat samples (vent 2 and vent 4) from the Calypso hydrothermal vent field, Bay of Plenty, New Zealand. Commonly usednirS andnirK primer sets nirS1F/nirS6R, cd3aF/R3cd, nirK1F/nirK5R, and F1aCu/R3Cu were also tested to determine what may be missed by the common single-primer approach to assessing denitrifier diversity. The relative importance ofEpsilonproteobacteria in these samples was evaluated by 16S rRNA gene sequencing.Epsilonproteobacteria represented up to 75.6% of 16S rRNA libraries, butnirS genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recoverednirS sequences from members of the generaSulfurimonas ,Sulfurovum , andNitratifractor. The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifyingEpsilonproteobacteria in vent 4 compared to vent 2, which had greater representation by “standard” denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly usednirK primers suggest biased amplification between primers. Future application of multiplenirS andnirK primers, including the new epsilonproteobacterialnirS primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities.IMPORTANCE Estimating the potential for increasing nitrogen limitation in the changing global ocean is reliant on understanding the microbial community that removes nitrogen through the process of denitrification. This process is favored under oxygen limitation, which is a growing global-ocean phenomenon. Current methods use the nitrite reductase genesnirS andnirK to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifyingEpsilonproteobacteria , a group known to dominate in reducing environments, such as hydrothermal vents and anoxic basins. As oxygen depletion expands in the oceans, it is important to study denitrifier community dynamics within those areas to predict future global ocean changes. This study explores the design and testing of new primers that target epsilonproteobacterialnirS and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.

Capturing Compositional Variation in Denitrifying Communities: a Multiple-Primer Approach That Includes Epsilonproteobacteria