Dataset Information

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.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 genes nirS and nirK to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifying Epsilonproteobacteria, 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 epsilonproteobacterial nirS and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.

SUBMITTER: Murdock SA

PROVIDER: S-EPMC5335531 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Murdock Sheryl A SA Juniper S Kim SK

Applied and environmental microbiology 20170302 6

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 Ple ...[more]

PMID: 28087525

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Project description:Background:The Hailuogou Glacier is located at the Gongga Mountain on the southeastern edge of the Tibetan Plateau, and has retreated continuously as a result of global warming. The retreat of the Hailuogou Glacier has left behind a primary succession along soil chronosequences. Hailuogou Glacier's retreated area provides an excellent living environment for the colonization of microbes and plants, making it an ideal model to explore plant successions, microbial communities, and the interaction of plants and microbes during the colonization process. However, to date, the density of the nitrogen cycling microbial communities remain unknown, especially for denitrifiers in the primary succession of the Hailuogou Glacier. Therefore, we investigated the structural succession and its driving factors for denitrifying bacterial communities during the four successional stages (0, 20, 40, and 60 years). Methods:The diversity, community composition, and abundance of nosZ-denitrifiers were determined using molecular tools, including terminal restriction fragment length polymorphism and quantitative polymerase chain reactions (qPCR). Results:nosZ-denitrifiers were more abundant and diverse in soils from successional years 20-60 compared to 0-5 years, and was highest in Site3 (40 years). The denitrifying bacterial community composition was more complex in older soils (40-60 years) than in younger soils (?20 years). The terminal restriction fragments (T-RFs) of Azospirillum (90 bp) and Rubrivivax (95 bp) were dominant in soisl during early successional stages (0-20 years) and in the mature phase (40-60 years), respectively. Specific T-RFs of Bradyrhizobium (100 bp) and Pseudomonas (275 bp) were detected only in Site3 and Site4, respectively. Moreover, the unidentified 175 bp T-RFs was detected only in Site3. Of the abiotic factors that were measured in this study, soil available phosphorus, available potassium and denitrifying enzyme activity (DEA) correlated significantly with the community composition of nosZ-denitrifiers (P < 0.05 by Monte Carlo permutation test within RDA analysis).

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Dataset Information

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

Publications

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

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