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Microbial Ecology

Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing

Boris Wawrik, Amy V. Callaghan, Deborah A. Bronk
Boris Wawrik
1Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma
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  • For correspondence: bwawrik@ou.edu
Amy V. Callaghan
1Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma
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Deborah A. Bronk
2 Virginia Institute of Marine Science, The College of William and Mary, Williamsburg, Virginia
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DOI: 10.1128/AEM.01002-09
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  • FIG. 1.
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    FIG. 1.

    Physical and chemical data collected at the study site on the west Florida shelf, including (A) photosynthetically active radiation (PAR) (□) and salinity (in practical salinity units [PSU]) (•) and (B) temperature (•) and dissolved oxygen concentration (□). All parameters are expressed as a function of depth.

  • FIG. 2.
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    FIG. 2.

    Phytoplankton community composition as determined by culture-independent methods. rbcL genes were amplified using two different (form IA/B- and form ID-specific) broad-range PCR primer sets, cloned, and sequenced. Sequences were grouped into operational taxonomic units with >95% sequence identity and compared to the GenBank database by means of BlastX searches. The charts show the phylogenetic distribution of the best BlastX matches for each of the operational taxonomic units recovered from station 9C. Phylum-level groups are enclosed in boxes. Class- and order-level groups are indicated by bold type. Genus-level taxonomic groups are in parentheses. (A) Pie chart indicating the frequencies of different types of form IA/B-like rbcL genes in the clone library. (B) Pie chart indicating the frequencies of different types of form ID-like rbcL genes in the clone library.

  • FIG. 3.
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    FIG. 3.

    Stable isotope incorporation showing 15NO3 uptake in a culture of Synechococcus sp. strain WH7803. (A) Relationship between buoyant density of DNA in a CsCl gradient, G+C content, and percentage of 15N incorporated. The solid diagonal line indicates the predicted buoyant density of DNA containing only 14N. The dashed line indicates the predicted buoyant density of DNA with 100% 15N incorporation. The horizontal line indicates the G+C content of WH7803, and the vertical lines indicate the predicted buoyant densities of WH7803 with 0% and 100% 15N incorporation. (B) Two cultures of Synechococcus sp. strain WH7803 were grown on 14NO3 and 15NO3 as N sources. DNA was extracted from both cultures, and both cultures were fractionated on CsCl gradients. The WH7803 DNA in each fraction was quantified by qPCR by determining the rbcL gene copy number in each 100-μl fraction. The data are the ratios of quantities, which were calculated by dividing the measured amount of Synechococcus rbcL DNA in each fraction by the highest value measured in any of the fractions collected from a CsCl column. ▴, unlabeled WH7803 DNA (0% 15N); •, labeled WH7803 DNA (100% 15N).

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    FIG. 4.

    Amount of Synechococcus DNA as a function of density as determined by CsCl centrifugation and quantitative PCR, using the Synechococcus rbcL gene as a proxy in field samples. The vertical lines correspond to the vertical lines in Fig. 3 and indicate the density of Synechococcus DNA containing only 14N or only 15N. (A) Data for the ambient population (T0) and for incubation without addition of a 15N-labeled nitrogen source (NTC). (B to F) Data obtained after incubation in the presence of 2 μmol N liter−1 of (B) [15N]urea, (C) 15NH4+, (D) 15NO3−, (E) 15N-amino acids (AA), and (F) [15N]glutamic acid (GA). The data are ratios of quantities, which were calculated by dividing the measured amount of Synechococcus rbcL DNA in each fraction by the highest value measured for any of the fractions collected from a CsCl column.

  • FIG. 5.
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    FIG. 5.

    Amount of diatom DNA as a function of density as determined by CsCl centrifugation and qPCR, using diatom rbcL genes as a proxy. The vertical lines correspond to the vertical lines in Fig. 1 and indicate the density of diatom DNA containing only 14N or only 15N. (A) Data for the ambient population (T0) and for incubation without addition of a 15N-labeled nitrogen source (NTC). (B to F) Data obtained after incubation in the presence of 2 μmol N liter−1 of (B) [15N]urea, (C) 15NH4+, (D) 15NO3−, (E) 15N-amino acids (AA), and (F) [15N]glutamic acid (GA). The data are ratios of quantities, which were calculated by dividing the measured amount of diatom rbcL DNA in each fraction by the highest value measured for any of the fractions collected from a CsCl column.

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  • TABLE 1.

    Ambient substrate concentrations and uptake and regeneration rates in surface water

    SubstrateConcn (μmol N liter−1)Uptake rate (μmol N liter−1 h−1)Regeneration rate (μmol N liter−1 h−1)
    NH4+ (ammonium)0.135 ± 0.0360.077 ± 0.0120.082 ± 0.008
    NO3− (nitrate)0.118 ± 0.0120.065 ± 0.0140.147 ± 0.035
    Urea0.328 ± 0.0670.013 ± 0.004NDa
    Dissolved primary amines (approximately amino acids)0.854 ± 0.2200.055 ± 0.001ND
    DON16.2 ± 0.271NDND
    PO4−3 (phosphate)0.028 ± 0.002NDND
    Dissolved organic phosphate0.635 ± 0.021NDND
    Biogenic silicate5.543 ± 0.374NDND
    • ↵ a ND, not determined.

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Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing
Boris Wawrik, Amy V. Callaghan, Deborah A. Bronk
Applied and Environmental Microbiology Oct 2009, 75 (21) 6662-6670; DOI: 10.1128/AEM.01002-09

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Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing
Boris Wawrik, Amy V. Callaghan, Deborah A. Bronk
Applied and Environmental Microbiology Oct 2009, 75 (21) 6662-6670; DOI: 10.1128/AEM.01002-09
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KEYWORDS

diatoms
Nitrogen Compounds
Nitrogen Isotopes
seawater
Synechococcus

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