Capturing compositional variation in denitrifying communities: A multiple-primer approach that includes epsilonproteobacteria

Abstract

Denitrifying Epsilonproteobacteria 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 the nirS gene of denitrifying Epsilonproteobacteria 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 used nirS and nirK 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 of Epsilonproteobacteria in these samples was evaluated by 16S rRNA gene sequencing. Epsilonproteobacteria represented up to 75.6% of 16S rRNA libraries, but nirS genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recovered nirS sequences from members of the genera Sulfurimonas, Sulfurovum, and Nitratifractor. The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifying Epsilonproteobacteria in vent 4 compared to vent 2, which had greater representation by "standard" denitrifiers measured with the cd3aF/ R3cd primers. Limited results from commonly used nirK primers suggest biased amplification between primers. Future application of multiple nirS and nirK primers, including the new epsilonproteobacterial nirS primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities.