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Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.

ABSTRACT: A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.

SUBMITTER: Braker G

PROVIDER: S-EPMC106545 | biostudies-literature | 1998 Oct

REPOSITORIES: biostudies-literature

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Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.

Braker G G Fesefeldt A A Witzel K P KP

Applied and environmental microbiology 19981001 10

A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory ...[more]

PMID: 9758798

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Project description:Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.

Project description:The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (encoding NO2-→NO reductases) and couples growth to NO3- reduction to NH4+ (respiratory ammonification) and to N2O reduction to N2A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO2- to form N2O (i.e., chemodenitrification). Following the addition of 100 μmol of NO3- or NO2- to Fe(III)-grown axenic cultures of A. dehalogenans, 54 (±7) μmol and 113 (±2) μmol N2O-N, respectively, were produced and subsequently consumed. The conversion of NO3- to N2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NCBI Reference Sequence database coupled with available physiological information revealed that organisms lacking nirS or nirK but with Fe(III) reduction potential and genes for NO3- and N2O reduction are not rare, indicating that NO3- reduction to N2 through linked biotic-abiotic reactions is not limited to A. dehalogenans Considering the ubiquity of iron in soils and sediments and the broad distribution of dissimilatory Fe(III) and NO3- reducers, denitrification independent of NO-forming NO2- reductases (through combined biotic-abiotic reactions) may have substantial contributions to N loss and N2O flux.IMPORTANCE Current attempts to gauge N loss from soils rely on the quantitative measurement of nirK and nirS genes and/or transcripts. In the presence of iron, the common soil bacterium Anaeromyxobacter dehalogenans is capable of denitrification and the production of N2 without the key denitrification genes nirK and nirS Such chemodenitrifiers denitrify through combined biotic and abiotic reactions and have potentially large contributions to N loss to the atmosphere and fill a heretofore unrecognized ecological niche in soil ecosystems. The findings emphasize that the comprehensive understanding of N flux and the accurate assessment of denitrification potential can be achieved only when integrated studies of interlinked biogeochemical cycles are performed.

Dataset Information

Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.

Publications

Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples.

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