Cordero Rodríguez, César E.

Profile Picture

Filters

20031
Reset filters

Settings

Citations

Publication Search Results

Now showing 1 - 1 of 1
  • No Thumbnail Available
    Publication
    Isolation, characterization and detection by 𝘪𝘯 𝘴𝘪𝘵𝘶 gene amplification of marine denitrifying bacteria
    (2003) Cordero Rodríguez, César E.; Massol-Deyá, Arturo A.; College of Arts and Sciences - Sciences; Castillo, Carlos; Diffoot, Nanette; Department of Biology; Schroder, Eduardo C.
    Recent studies suggest that the oceanic nitrogen budget is unbalanced, primarily due to a high nitrogen removal in contrast to the fixation rate. This imbalance likely results from denitrification activity in continental shelf sediments. Denitrifying bacteria play a major role in marine sediment nitrogen balance. In order to assess the nitrogen balance characteristic, this study utilized slow-growth enrichment microcosms. These consisted of Puget Sound, WA seawater and sediment samples enriched with dimethyl sulfoxide (DMSO) as carbon source, and nitrate in order to stimulate denitrifying activity. Two sets of microcosms were prepared and incubated in the dark for 6 months at 25oC and 4oC. Of 82 strains isolated, 18 were positive for both nitrate reduction and gas production. Amplified ribosomal DNA restriction analysis (ARDRA) was performed to compare and establish similarities between the Puget Sound isolates and control cultures. The novel in situ reverse transcription PCR (RT-PCR) method has been optimized to study the expression of the nirS gene in denitrifying bacteria. Pure cultures of reference denitrifying isolates from marine sediments were used to optimize the in situ RT-PCR protocol. We performed cell fixation after visible gas production was observed. In situ RT-PCR was performed after cell fixation and enzymatic permeabilization. The nirS 1F and nirS 6R primers were used for the amplification of cDNA and subsequently fluorescent in situ hybridization (FISH) was done to increase the detection specificity of the amplification product. Only active denitrifying cells were detected by this approach using fluorescence microscopy.