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

Sheryl A Murdock1, S Kim Juniper2,3

  • 1School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada smurdock@uvic.ca.

Applied and Environmental Microbiology
|January 15, 2017
PubMed
Summary

New primers targeting nitrite reductase genes (nirS) were developed for Epsilonproteobacteria, crucial for nitrogen loss in marine environments. These new primers improve the detection of denitrifying Epsilonproteobacteria, enhancing our understanding of nitrogen cycling in changing oceans.

Keywords:
EpsilonproteobacteriadenitrificationhydrothermalnirSprimers

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Area of Science:

  • Marine microbiology
  • Biogeochemical cycles
  • Molecular ecology

Background:

  • Denitrifying Epsilonproteobacteria play a key role in marine nitrogen loss, particularly in redox gradients.
  • Current methods using nitrite reductase genes (nirS and nirK) often miss these important bacteria due to primer limitations.
  • Understanding denitrifier communities is crucial for predicting nitrogen limitation in the changing global ocean.

Purpose of the Study:

  • To design and test new primers targeting the nirS gene of denitrifying Epsilonproteobacteria.
  • To evaluate the effectiveness of commonly used nirS and nirK primers in detecting denitrifier diversity.
  • To assess the relative abundance of Epsilonproteobacteria in marine hydrothermal vent samples.

Main Methods:

  • Design and validation of nine new nirS primers specific to denitrifying Epsilonproteobacteria.
  • Application of new and existing nirS and nirK primers in sequencing and quantitative PCR.
  • 16S rRNA gene sequencing to determine the relative abundance of Epsilonproteobacteria.

Main Results:

  • Epsilonproteobacteria constituted up to 75.6% of 16S rRNA sequences in the studied microbial mats.
  • Commonly used nirS primers failed to detect nirS genes from Epsilonproteobacteria.
  • New primers successfully amplified nirS sequences from Epsilonproteobacteria genera like Sulfurimonas, Sulfurovum, and Nitratifractor.
  • Quantitative PCR revealed higher abundance of denitrifying Epsilonproteobacteria in vent 4 compared to vent 2.

Conclusions:

  • Existing nirS and nirK primers are insufficient for comprehensive denitrifier diversity assessment, systematically excluding Epsilonproteobacteria.
  • The newly developed epsilonproteobacterial nirS primers are essential for accurately quantifying these key denitrifiers.
  • A multiple-primer approach, incorporating these new primers, is recommended for improved detection of denitrifier diversity and community dynamics.