Detection and diversity of copper containing nitrite reductase genes (nirK) in prokaryotic and fungal communities of agricultural soils

Long, Andrew; Song, Bongkeun; Fridey, Kelly; Silva, Amy

doi:10.1093/femsec/fiu004

Cited by 41 publications

(29 citation statements)

References 63 publications

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“…To date, only a limited number of studies have relied on molecular methods to detect, identify, and/or characterize fungal denitrifiers (13,27,28,29,35,36). The study by Long et al (36) (4,17,37), most strains isolated in the present study were not able to reduce NO 3 Ϫ to N 2 O, with the exception of Fusarium strains (data not shown).…”

Section: Discussionmentioning

confidence: 52%

“…The mismatch at this site was not observed in the 27 P450nor sequences determined in this study and may offer an explanation as to why P450nor sequences from C. rosea were not amplified. The nirK primers developed by Maeda et al (13), Long et al (28), and Wei et al (27) were also used to attempt to amplify the nirK gene from the eight strains of C. rosea using their respective PCR protocols; however, bands of the correct size were not observed for any of the primer pairs used in the eight C. rosea strains (results not shown). The P450nor PCR primers and nirK PCR primers developed in this study detected most denitrifying species tested in the current study, but they show some limitations, like most PCR primer sets targeting functional microbial genes, being incapable of generating amplicons from at least one genus of known fungal denitrifiers, Clonostachys.…”

Section: Discussionmentioning

confidence: 99%

“…With little attention having been focused on denitrifying fungi in soil, their abundance and diversity are not well documented. Molecular-based methods have been developed and extensively used to target bacterial denitrifiers; however, analogous tools have only recently been developed for fungal denitrifiers (13,27,28,29). In this context, the P450nor gene appears to be a better molecular target for detecting and characterizing fungal denitrifiers due to the lack of homology existing between the bacterial cnorBqnorB and fungal P450nor genes (14).…”

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confidence: 99%

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Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Novinscak

Zebarth

Burton

et al. 2016

Appl Environ Microbiol

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Denitrifying fungi produce nitrous oxide (N 2 O), a potent greenhouse gas, as they generally lack the ability to convert N 2 O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N 2 O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N 2 O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N 2 O from nitrite. N 2 O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developedP450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N 2 O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor. The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

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Section: Discussionmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Novinscak

Zebarth

Burton

et al. 2016

Appl Environ Microbiol

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“…Amplification of fungal nirK genes from isolate DNA were performed as previously described by using primers nirKfF/nirKfR and fnirK2F/ fnirK1R (34,35) with the following modifications. The durations of the annealing and elongation steps were increased by 15 s (34) or decreased by 15 s (35), respectively, to allow simultaneous amplification of similar-size amplicons (ϳ450 to 500 bp).…”

Section: Medium Preparationmentioning

confidence: 99%

“…For amplification of p450nor from soil DNA templates, as well as amplification of isolate ITS regions or isolate 18S rRNA genes, the annealing temperature was increased to 50, 53, or 55°C, respectively. Annealing temperatures for amplifications with nirK-targeted primer pairs nirKfF/nirKfR and fnirK2F/fnirK1R were 54 and 50°C, respectively (34,35). For visualization of amplicons, 5 l of the PCR assay mixture was combined with 1 l of a loading dye solution (25 mg each bromophenol blue and xylene cyanol and 4 g sucrose dissolved in 10 ml of ultrapure water) and loaded onto a 1% (wt/vol) agarose gel, and electrophoresis was conducted at 90 V for 1 h at an initial amperage of 200 mA.…”

Section: Medium Preparationmentioning

confidence: 99%

Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Higgins

Welsh

Orellana

et al. 2016

Appl Environ Microbiol

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Members of the Fungi convert nitrate (NO 3؊ ) and nitrite (NO 2 ؊ ) to gaseous nitrous oxide (N 2 O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2 O from added NO 3 ؊ or NO 2 ؊ in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N 2 O from NO 2 ؊ . Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N 2 O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2 O from NO 2 ؊ , whereas nirK (encoding the NO-forming NO 2 ؊ reductase) was amplified in only 13 to 74% of the N 2 O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2 O formation. IMPORTANCEA comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N 2 O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N 2 O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N 2 O emissions. Denitrification is a key process responsible for loss of fixed nitrogen (N) in soils and sediments and is mediated by both abiotic and microbial processes (1, 2). Of the microorganisms involved in denitrification, members of the Bacteria are well studied and considered key contributors; however, some saprotrophic fungi also conserve energy from the reduction of nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) to nitrous oxide (N 2 O), the main end product of fungal denitrification (3-5). Fungi have been implicated in N turnover for over 30 years (1,3,6), but the ecological importance of fungal contributions to denitrification remain uncertain. Potential roles of fungi in soil N loss and greenhouse gas (i.e., N 2 O) emission are underexplored, and monitoring tools (e.g., quantitative or endpoint PCR) to specifically address the presence and abundance of denitrif...

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Copper‐Containing Nitrite Reductase

Rose

Hough

Antonyuk

et al. 2022

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Cu‐containing nitrite reductases (CuNiRs) catalyze the reduction of nitrite to nitric oxide and water. CuNiRs are part of the denitrification and nitrification pathways that are not only important from bioenergetics perspective but are also the major contributors to terrestrial and oceanic nitrogen cycling. These pathways make a significantly increasing contribution to global warming by release of N 2 O, an ozone‐depleting and greenhouse gas some 300‐fold more potent than CO 2 . Advanced crystallographic techniques based on synchrotrons and X‐ray free electron lasers have been applied to this important class of enzymes. Here we provide a detailed review of recent results obtained from a global effort.

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Detection and diversity of copper containing nitrite reductase genes (nirK) in prokaryotic and fungal communities of agricultural soils

Cited by 41 publications

References 63 publications

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Copper‐Containing Nitrite Reductase

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