Mary F. DeFlaun

Particulate DNA in subtropical oceanic and estuarine planktonic environments

Particulate DNA was measured in estuarine, coastal, and oligotrophic oceanic environments near the southwest coast of Florida and in the Gulf of Mexico. Particulate organic carbon and nitrogen (POC and PON), chlorophyll a, bacterial direct counts, DNA, and bacterial activity as determined by thymidine incorporation all showed a high degree of intercorrelation. Normalization of the data for offshore/onshore similarities by dividing by POC yielded significant correlations only for DNA, direct counts, and bacterial activity. Most (70–99%) of the particulate DNA in offshore samples was in the 0.2- to 1-μm fraction, while DNA in nearshore and estuarine samples was associated with larger particles. Cellular DNA contents obtained by dividing DNA by direct counts in the 0.2- to 1-μm fraction were in the range of reported bacterial genome weights. However, DNA nitrogen comprised a greater proportion of the PON than reported for microorganisms in culture. Collectively, these results suggest that (1) most of the particulate DNA in oceanic environments is contained in bacterioplankton; (2) DNA is a significant proportion of the cell biomass, possibly due to growth under nutrientlimiting conditions.

Detection of exogenous gene sequences in dissolved DNA from aquatic environments.

A method for the concentration and detection of gene sequences in the dissolved DNA from freshwater and marine environments has been developed. The limit of detection in the dot blot format was 167 fg/ml (100 ml sample) for exogenous herpes simplex thymidine kinase (TK) gene that was added to artificial seawater or river water. This procedure has been used to determine the longevity and monitor progressive changes in molecular weight of a plasmid containing the TK gene added to eutrophic estuarine water. The onset of plasmid degradation as determined by change in molecular weight was rapid (within 5 min). Intact plasmid was detected for at least 4 hours and sequences hybridizable to the TK gene probe were present for up to 24 hours.

Hoechst 33258 staining of DNA in agarose gel electrophoresis

Abstract A staining procedure for agarose gel electrophoresis using Hoechst 33258 has been optimized. A concentration of 5 × 10−8 M allows the immediate visualization of as little as 1 ng of DNA with a standard UV illumination system. The major advantage of this method is the elimination of the carcinogen ethidium bromide used in most DNA visualization methods for gel electrophoresis.

The distribution of dissolved DNA in an oligotrophic and a eutrophic river of Southwest Florida

The distribution of dissolved DNA concentrations and some microbial variables were compared in an oligo-mesotrophic river (the Crystal River) and a phosphate-rich eutrophic river (the Alafia River) in Southwest Florida over a 15 month period. Concentrations of phosphate and nitrate in the Alafia River averaged 135 and 18.2 times the respective phosphate and nitrate concentrations of the oligo-mesotrophic Crystal River. The seasonal average dissolved DNA concentration for the Alafia River exceeded that of the Crystal River by a factor of 1.8 (8.2 μg 1−1 compared to 4.6 μg 1−1, respectively). The greatest concentrations of dissolved DNA in the Alafia River were found in areas that contained the largest populations of phytoplankton and bacteria (a reservoir formed from an abandoned phosphate mining pit and two downstream stations near the mouth of the river). Differences in dissolved DNA concentrations between these environments and more pristine environments (i.e. all Crystal River Stations and upstream Alafia River stations) were of the same order of magnitude (1.8 to 2.2-fold) as the differences in bacterial abundance and activity, but considerably less than differences in phytoplankton abundance and activity between such environments. Seasonal variations in dissolved DNA concentrations in the Crystal River corresponded to seasonal variations in microbial populations, with minimal values in January and greater values in July. In the Alafia River, lowest concentrations for dissolved DNA occurred in July during the wet season, when seasonal flooding of area of leaf litter yielded high levels of dissolved organic carbon (DOC) which were low in dissolved DNA. These results suggest that: 1) in situ planktonic activity is a greater source of dissolved DNA than allochthonous or terrestrial sources of DOC; 2) factors that control the magnitude of heterotrophic bacterial populations are more likely to control dissolved DNA levels than factors regulating autotrophic population activity and abundance; 3) differences in dissolved DNA between eutrophic and oligo-mesotrophic environments are often much smaller than the differences in nutrient concentration between such environments.

Correlation of nonspecific macromolecular labeling with environmental parameters during [3H]Thymidine incorporation in the waters of southwest florida

AbstractDuring routine [3H]thymidine incorporation measurements of environmental samples, significant amounts of radioactivity are often incorporated into macromolecules other than DNA. Although the percentage of nonspecific labeling varies both temporally and spatially, the cause(s) of these variations remain unknown. Correlations between the percent incorporated radioactivity in DNA and a variety of experimental and environmental parameters measured in the Alfia River, Crystal River, Medard Reservoir, and Bayboro Harbor were examined. The amount of radioactivity incorporated into DNA ranged from 6 to 95% (n

Elevated Levels of Microbial Activity in the Coral Surface Microlayer

bar x = 51 pm 14%

Turnover of extracellular DNA in eutrophic and oligotrophic freshwater environments of southwest Florida

n; n=121). Nonspecific labeling began immediately upon the addition of [3H]thymidine and was linear over time. Labeling patterns were independent of both the amount of thymidine added and cell-size fraction. A two year study of Bayboro Harbor indicated no conclusive relationship between nonspecific labeling and seasonality. The amount of radioactivity incorporated into DNA was inversely correlated with total rates of thymidine incorporation and a strong diurnal pattern was observed in the Crystal River. No consistent relationship was observed between labeling patterns and primary productivity, chlorophylla, particulate DNA, dissolved DNA, bacterial cell numbers, temperature, salinity, and dissolved organic carbon. The only relationship with dissolved inorganic nutrients (N and P) occurred in the Crystal River. In this phosphate limited river, the percent of radioactivity incorporated into DNA was positively correlated with phosphate concentrations. These results indicate that nonspecific labeling is not dependent on any one parameter but may be a function of many interacting environmental factors or a function of the specific ambient bacterial population.