Efficiency of DNA extraction with the QBiogene FastDNA system

The coupling between the presence of predominant bacterial species and particular biogeochemical processes is of primary interest in current microbial ecology. Molecular methods such as quantitative real-time polymerase chain reaction (PCR) may be used to estimate presence or expression of different genes (e.g., 16S rDNA for abundance of certain bacteria groups, nifH genes for presence or activity of nitrogen-fixing cyanobacteria, etc.) in environmental samples. However, to be quantitative, these methods require a reproducible and efficient DNA extraction protocol (Boström et al. 2004). A wide variety of DNA extraction methods are currently in use for aquatic community analyses. One of the original extraction methods (Fuhrman et al. 1988) was recently optimized for marine bacteria, reaching an extraction efficiency (i.e. ratio of DNA recovered from a sample after extraction to DNA in the original sample) of 92-96% (Boström et al. 2004), in contrast to efficiencies of 20-60% in the original protocol and other methods (Fuhrman et al. 1988, Weinbauer et al. 2002). However, this optimized, classical method still involves many manual steps for DNA extraction. Modern, commercial extraction kits offered by various manufacturers offer easy and much faster DNA extraction, usually through "spin-filter". In addition to faster processing, use of these spin-filter kits promise the advantage of reduced contamination risk during extraction procedures, cleaner resulting DNA extracts (i.e. less protein contamination), higher reproducibility, and more efficient removal of contaminants potentially inhibiting the PCR reaction, for which the DNA extracts are usually produced. The problem of PCR-inhibitors is particularly severe in biological samples from seawater and marine sediments. The QBiogene FastDNA Spin Kit for Soil is said to show particularly high efficiency in the removal of such PCR inhibitors and to exhibit very high reproducibility among replicate samples. However, DNA extraction efficiency of this promising spin kit for quantitative aquatic work has not been assessed yet. The undergraduate summer study will assess the efficiency of bacterial DNA extraction of the QBiogene FastDNA Spin Kit for Soil following an experimental design outlined by Boström et al. (2004).

Boström KH, Simu K, Hagström A, Riemann L (2004) Optimization of DNA extraction for quantitative marine bacterioplankton community analysis. Limnol. Oceanogr. Methods 2: 365-373.
Fuhrman JA, Comeau DE, Hagström A, Chan AM (1988) Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Appl. Environ. Microbiol. 54: 1426-1429.
Weinbauer MG, Fritz I, Wenderoth DF, Höfle MG (2002) Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses. Appl. Environ. Microbiol. 68: 1082-1087.

Linking gene expression to biogeochemical processes:
Relation of amoA gene expression and ammonium oxidation rates in nitrifying bacteria

Bacteria play an important role in the biogeochemical cycling of nitrogen in marine environments. Not only are bacteria responsible for the degradation of dead, particulate organic carbon (detritus) and dissolved organic carbon (DOC), thereby excreting part of the nitrogen contained in the detritus or DOC as NH4⁺, which then in turn is available for phytoplankton utilization. Certain groups of bacteria also convert one inorganic form of nitrogen into another. Nitrifying bacteria convert NH4⁺ into NO2^- (ammonium oxidizing bacteria), and subsequently NO2^- into NO3^- under oxic conditions. Denitrifying bacteria perform a reverse transformation, converting NO3^- into gaseous forms (N2 or N2O), which will lead to a net loss of dissolved nitrogen in the aquatic ecosystem. In some benthic systems, NO3^- is not denitrified but converted to NH4⁺ by dissimilatory nitrate reduction to ammonium (DNRA). Bacterial transformations might not only be important for the flow of nitrogen through the ecosystem. High rates of nitrification have been hypothesized to be related to the formation of oxygen minimum zones or increasing hypoxia, for example in the Arabian Sea and the Mississippi River plume in the Gulf of Mexico (Pakulski et al. 1995, Ward 2000). Quantification of biogeochemical nitrogen transformations is less than straightforward, though (Ward 2000), and requires sophisticated techniques and instrumentation (e.g. mass spectrometer for ¹⁵N stable isotope tracer experiments, membrane inlet mass spectrometer (MIMS) for measurement of denitrification, nitrogen fixation, and/or DNRA) available at only few labs. Nitrification rates were assessed directly by incubation methods employing either specific inhibitors of nitrification and following the accumulation of NO2^- , or utilizing the chemoautotrophic nature of nitrifying bacteria (i.e. they acquire their carbon need through CO 2) and measuring radiolabeled ¹⁴CO2 uptake in the dark. These assays are, however, not without problems (Ward 2000). For example, accumulation of NO2^- as a result of nitrification (ammonium oxidation) might not be detectable or underestimated in natural, mixed populations because produced NO2^- might be consumed instantaneously; a decrease in NH4⁺ concentrations, even when incubating samples in the dark, might not be caused by ammonium oxidizing bacteria alone but by the uptake by phytoplankton and heterotrophic bacteria. Meanwhile, molecular techniques have revealed the genetic sequence of the amoA gene that encodes the enzyme ammonia monoxygenase, which catalyzes the oxidation of NH4⁺ to NO2 ^- Sequence analysis has revealed relatively little genetic variation in the amoA gene, and nitrifying bacteria in general (Ward 2000). Given the difficulties in quantifying aquatic biogeochemical nitrogen transformations directly, assessing these processes by quantifying gene expression of responsible genes appears a promising venue. Before such techniques can be applied to natural systems and populations, it has to be demonstrated that gene expression levels are, in fact, good predictors of biogeochemical, metabolic rates. This undergraduate summer project aims at measuring amoA gene expression in nitrifying bacteria isolated from Biscayne Bay marine sediments under different growth conditions by quantitative reverse-transcription real-time PCR. In parallel, the decrease in NH 4⁺ and the increase in NO2^- will be measured to quantify nitrification (ammonium oxidation) rates in the bacterial cultures, which will be related to ambient amoA gene expression.

Pakulski J D, Benner R, Amon R, Eadie B, Whitledge T (1995) Community metabolism and nutrient cycling in the Mississippi River plume: evidence for intense nitrification at intermediate salinities. Mar. Ecol. Prog. Ser. 117: 207-218.
Ward BB (2000) Nitrification and the marine nitrogen cycle. In: Kirchman DL [ed]: Microbial Ecology of the Oceans. Wiley-Liss, New York. 427-453.

Immunolabeling of a cell division-specific nuclear protein in phytoplankton - optimization for application in flow cytometry

Estimates of intrinsic growth rates of phytoplankton and/or single species of phytoplankton in mixed natural populations under natural conditions remains a challenging task in biological oceanography. However, reliable estimates of phytoplankton growth rates are essential in evaluating effects of environmental changes (e.g. global warming, eutrophication) on natural phytoplankton communities. Current techniques include measuring primary production by radioactive ¹⁴CO2 uptake incubations or so-called serial dilution experiments. While widely applied, both techniques have certain drawbacks (e.g. radioisotope use) and require shorter or longer bottle incubations and manipulations, which in themselves might affect phytoplankton growth. Proliferating Cell Nuclear Antigen (PCNA) is a protein only expressed during the S-phase of the eukaryote cell cycle, i.e. the phase of the cell cycle during which DNA is replicated in preparation for mitosis and cell division. Dr. Senjie Lin (University of Connecticut) has shown in the past that specific antibodies against PCNA can be used to specifically label cells within a population that currently undergo DNA replication (S-phase cells). The specific PCNA antibody is either fluorescently labeled directly, or a secondary antibody against the PCNA antibody carries a fluorescent dye, which allows detection upon binding of the secondary antibody with the primary PCNA antibody. Cells can be observed under a fluorescence microscope to detect positive labeling. Within an NSF-funded project and in collaboration with Dr. Lin, PCNA immunolabeling shall be optimized for application in high-throughput analytical flow cytometry. This summer project will use samples from different growth experiments of the green alga Dunaliella tertiolecta and PCNA antibodies developed for this species (both provided by Dr. Lin) for flow cytometry protocol development and optimization. Results from PCNA immunolabeling will be compared to direct measurements of the cells' cell cycle phase by cytometric quantification of cellular DNA content. The planned summer project is an important stepping stone for the progress of this NSF project.

Genetic population structure of deep-sea gulper sharks and Cuban Dogfish
from the Gulf of Mexico

During a student research and training course (OCB5993 Oceanography at Sea) in April 2005 at the shelf break off St. Petersburg, FL, gulper sharks and Cuban Dogfish were caught by longline fishing between 800 and 1200 m depth. This effort represents the first in a planned series of efforts and cruises to collect deep-sea sharks at different locations around the Florida peninsula to assess their genetic variability and genetic population structure. Comparison of genetic population structure will eventually allow assessing spreading patterns, or geographic isolation, of deep-sea shark populations around Florida. This information will provide helpful and new insight into the population ecology of hitherto largely understudied deep-sea sharks. As a first step in this multi-year effort, specimen collected in April shall be examined on their genetic profiles. We will use genetic fingerprinting techniques for mitochondrial DNA (mtDNA) and nuclear DNA. While mtDNA fingerprinting will reveal maternal effects only (mtDNA originates only from the mother), nuclear DNA fingerprinting will reveal combined maternal and paternal effects and relationships among the specimen caught in close vicinity in the Gulf of Mexico. Involved techniques include DNA extractions, PCR (polymerase chain reaction) amplification of diagnostic segments of nuclear DNA, DGGE (Denaturing Gradient Gel Electrophoresis) to fingerprint amplified nuclear DNA fragments, and multiple restriction digestion for mtDNA fingerprinting. Thus, this project will provide intensive training in modern molecular techniques. A minimum experience in lab work (e.g. precise pipetting, clean work) is beneficial.