title: Capturing Compositional Variation in Denitrifying Communities: a Multiple-Primer Approach That Includes Epsilonproteobacteria.
abstract: DenitrifyingEpsilonproteobacteriamay 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 thenirSgene of denitrifyingEpsilonproteobacteriawere 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 usednirSandnirKprimer 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 ofEpsilonproteobacteriain these samples was evaluated by 16S rRNA gene sequencing.Epsilonproteobacteriarepresented up to 75.6% of 16S rRNA libraries, butnirSgenes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recoverednirSsequences from members of the generaSulfurimonas,Sulfurovum, andNitratifractor.The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifyingEpsilonproteobacteriain vent 4 compared to vent 2, which had greater representation by "standard" denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly usednirKprimers suggest biased amplification between primers. Future application of multiplenirSandnirKprimers, including the new epsilonproteobacterialnirSprimers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities.IMPORTANCEEstimating 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 genesnirSandnirKto 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 epsilonproteobacterialnirSand reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.
authors.affiliation: School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada smurdock@uvic.ca.
authors.forename: Sheryl A
authors.initials: SA
authors.lastname: Murdock
authors.affiliation: School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada.|Department of Biology, University of Victoria, Victoria, British Columbia, Canada.
authors.forename: S Kim
authors.initials: SK
authors.lastname: Juniper
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D058440:Denitrification / Q000235:genetics* / Q000502:physiology; D017753:Ecosystem; D020565:Epsilonproteobacteria / Q000235:genetics* / Q000378:metabolism*; D019015:Geologic Sediments / Q000382:microbiology*; D060086:Hydrothermal Vents / Q000382:microbiology*; D009520:New Zealand; D009572:Nitrite Reductases / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D009792:Oceans and Seas; D010100:Oxygen / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D009572:Nitrite Reductases; D010100:Oxygen
keywords: Epsilonproteobacteria; denitrification; hydrothermal; nirS; primers