Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (<i>nirK</i>and<i>nirS</i>) To Detect Denitrifying Bacteria in Environmental Samples

Braker, Gesche; Fesefeldt, Andreas; Witzel, Karl-Paul

doi:10.1128/aem.64.10.3769-3775.1998

Cited by 758 publications

(295 citation statements)

References 35 publications

(45 reference statements)

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“…For the amplification of nirS gene, nirS1F (CCTA(C/T) T GGCCG CC (A/G) CA (A/G) T) and nirS6R (CGTTGAACTT(A/G)CCGGT) primer systems were used (Braker, Fesefeldt & Witzel, 1998 (Tamura et al, 2011). The evolutionary history was inferred using the UPGMA method (Sneath & Sokal, 1973), and the evolutionary distances were computed using the Kimura two-parameter method (Kimura, 1980).…”

Section: Phenotypic Characterization Of the Isolatesmentioning

confidence: 99%

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

et al. 2017

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Nitrate reducing potency of 88 bacterial isolates segregated from marine and brackish water nitrifying bacterial consortia (NBC), generated for activation of nitrifying bioreactors, was confirmed by determining the nitrate reducing capability under aerobic conditions as maintained in nitrifying bioreactors. All the isolates had the potential to be used as bio‐augmentors for activating nitrate dissimilation in recirculating aquaculture system. The existence of nitrate reducers with nitrifiers in NBC and in the reactor configuration negates the requirement of integrating anoxic denitrifying system for effective removal of NO3−‐N. Phylogenetic analyses of representative isolates from each cluster of the dendrograms generated based on phenotypic characterization and amplified ribosomal DNA restriction analysis revealed profound diversity of nitrate reducing bacterial flora in the NBC. They were composed of Streptomyces enissocaesilis, Marinobacter sp., Pseudomonas sp., Microbacterium oxydans, Pelagibacterium halotolerans and Alcanivorax dieselolei from marine NBC and Streptomyces tendae, Nesterenkonia sp., Bacillus cereus, Microbacterium oxydans and Brevibacterium sp. from brackish water NBC. The diversity indices of the consortia were calculated using Mega 5.0, primer 7 and VITCOMIC softwares. Marine NBC exhibited higher Shannon wiener diversity and mean population diversity than brackish water NBC. The study delineated higher species richness and diversity in marine NBC than in its brackish water counterpart, a possible reflection of the higher biodiversity of marine systems, and hence, the former is more promising to be used as start‐up cultures for the activation of nitrifying bioreactors after appropriate acclimatization to the desired salinity.

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Section: Phenotypic Characterization Of the Isolatesmentioning

confidence: 99%

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

et al. 2017

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“…Primers used for nirK quantification were nirK-q-F (5′-TCATGGTGCTGCCGCGYGA, a shorter version of the nirK583FdegCF forward primer from Santoro et al, 2006) and nirK1040 (Henry et al, 2004). Primers used for nirS quantification were nirS1F (Braker et al, 1998) and nirS-q-R (5′-TCCMAGCCRCCRTCRTGCAG), an upstream and significantly modified version of nirS3R (Braker et al, 1998).…”

Section: Quantification Of Nirs and Nirk Gene Abundancementioning

confidence: 99%

“…Total community DNA was extracted as described above. Sediment DNA extracts were PCR amplified with primers targeting nirK and nirS: nirK583FdegCF (5′-TCATGGTGCTGCCGCGYGANGG; Santoro et al, 2006) and nirK5R (Braker et al, 1998); nirS1F and nirS6R (Braker et al, 1998). Clone libraries of nirK and nirS gene fragments were constructed using the TOPO TA cloning ® kit (Invitrogen) and 28-45 clones from each library were sequenced (ABI 3100 Capillary Sequencer).…”

Section: Denitrifier Community Compositionmentioning

confidence: 99%

Denitrifier abundance and activity across the San Francisco Bay estuary

Mosier

Francis²

2010

Environ Microbiol Rep

120

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Over 50% of external dissolved inorganic nitrogen inputs to estuaries are removed by denitrification - the microbial conversion of nitrate to nitrogen gas under anaerobic conditions. In this study, denitrifier abundance, potential rates and community structure were examined in sediments from the San Francisco Bay estuary. Abundance of nirK genes (encoding Cu-containing nitrite reductase) ranged from 9.7 × 10(3) to 4.4 × 10(6) copies per gram of sediment, while the abundance of nirS genes (encoding cytochrome cd1 nitrite reductase) ranged from 5.4 × 10(5) to 5.4 × 10(7) copies per gram of sediment. nirK gene abundance was highest in the riverine North Bay, whereas nirS gene abundance was highest in the more marine Central and South Bays. Denitrification potential (DNP) rate measurements were highest in the San Pablo and Central Bays and lowest in the North Bay. nirS-type denitrifiers may be more biogeochemically important than nirK-type denitrifiers in this estuary, because DNP rates were positively correlated with nirS abundance, nirS abundance was higher than nirK abundance at every site and time point, and nirS richness was higher than nirK richness at every site. Statistical analyses demonstrated that salinity, organic carbon, nitrogen and several metals were key factors influencing denitrification rates, nir abundance and community structure. Overall, this study provides valuable new insights into the abundance, diversity and biogeochemical activity of estuarine denitrifying communities and suggests that nirS-type denitrifiers likely play an important role in nitrogen removal in San Francisco Bay, particularly at high-salinity sites.

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“…DNA from soil samples was extracted according to a cell lysis protocol involving bead beating as described previously (Peng et al, 2008;Qiu et al, 2008). PCR and T-RFLP of nirK genes were performed as described in Bremer et al (2007) using primer pair of nirK1F and nirK5R (Braker et al, 1998) with the reverse primer labelled with FAM and HaeIII (TaKaRa) as restriction enzyme.…”

Section: Dynamics Of Nirs Communitymentioning

confidence: 99%

Differential responses of nirK‐ and nirS‐carrying bacteria to denitrifying conditions in the anoxic rice field soil

Yuan¹,

Liu²,

Lu³

2011

Environ Microbiol Rep

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Denitrification occurs actively in rice field soils. In the present study, the responses of nirK and nirS denitrifier communities to nitrate addition in the anoxic rice soil were determined through molecular analyses of nitrite reductase genes nirK and nirS and 16S rRNA genes. Denitrification occurred rapidly when nitrate was added at the beginning of anoxic incubation (experiment I). The structure of nirK-type denitrifiers did not change; but their abundance as determined by quantitative (real-time) PCR increased in nitrate treatments compared with control. Both the structure and abundance of nirS denitrifiers remained unaffected in experiment I. The rate of denitrification was slowed down when nitrate was added 20 days after the onset of anoxic incubation (experiment II). The structure and abundance of nirK-type denitrifier community did not respond to nitrate addition; but the nirS community changed substantially in this experiment. The copy number of nirS genes increased by an order of magnitude in the treatments of 5 mM and 10 mM nitrate compared with control. The terminal restriction fragment length polymorphism (T-RFLP) analysis of nirS genes revealed that the 100 bp T-RF substantially increased in the nitrate treatments. Cloning and sequence analysis indicated that this T-RF had similarity of up to 90% with Herbaspirillum sp. T-RFLP profiles of the bacterial 16S rRNA genes also showed that Herbaspirillum sp. increased after nitrate amendments. Collectively, the nirK-type denitrifiers were probably active at the beginning of anaerobic incubation, while the nirS denitrifiers, especially those related with Herbaspirillum sp. probably were more active when anaerobic condition was fully developed.

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Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirKandnirS) To Detect Denitrifying Bacteria in Environmental Samples

Cited by 758 publications

References 35 publications

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

Genetic diversity of nitrate reducing bacteria in marine and brackish water nitrifying bacterial consortia generated for activating nitrifying bioreactors in recirculating aquaculture systems

Denitrifier abundance and activity across the San Francisco Bay estuary

Differential responses of nirK‐ and nirS‐carrying bacteria to denitrifying conditions in the anoxic rice field soil

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