For my Summer research this year, I applied for the DeFontes Grant. To essentially summarize the research I’m doing this Summer, my grant proposal follows. The focus has shifted more to the Outer Banks study, and my work with community profiling is centering on PFGE.

Efficient Methods for the Study of Viruses in a Soil Community

Introduction

For many years, soils have been the focus of studies in microbial ecology. From antibiotics to agronomy, many facets of the soil microbial community have been studied. This research has proven very important in the realms of medicine and ecology. One aspect of the soil community, however, has been largely neglected: viruses. In recent years, the research on soil viruses has expanded, but much work remains to be done.

Though the research on viruses in marine environments has been far more extensive, the importance of soil virus research cannot be overlooked. We lack refined techniques and procedures for extraction of viruses from soil samples, as well as efficient means of determining diversity from these extractions. As a result, our research on the bigger picture of soil community structure has been limited and incomplete. My research for the summer will focus on expanding these techniques, as well as furthering the study of the soil community. Specific objectives are to:

  1. Develop efficient methods for viral extraction from soil
  1. Measure viral diversity in order to track changes in the community structure over time and space
  2. Study the community structure of viruses in a coastal gradient

Background

Viruses have been studied fairly extensively in marine ecosystems. Research has determined that viruses play an important role in these communities, affecting the diversity and abundance of their hosts. Through lysis of host cells, viruses affect biogeochemical pathways in aquatic systems by the release of carbon and nitrogen. Viruses also drive host evolution through selection for resistant hosts and horizontal gene transfer. Given the very high abundance of viruses in soil, it is logical to assume that viruses play a comparably important role in the soil environment to viruses in the marine environment.

By utilizing field sites on our own campus – Lake Matoaka – we can begin to understand more about the soil community at large, as well as the interactions between the soil and aquatic communities. The work done this Summer will contribute to a more extensive on-going study regarding temporal dynamics of the viral community in Lake Matoaka and its watershed.

Objective 1: Develop efficient methods for viral extraction from soil

As we continue our work with soil viruses, it is beneficial to explore the most efficient options for the extraction of virus particles. There are three main techniques the study will focus on: sonication; blending; and bead-beating. These methods all break apart soil aggregates and dissociate viruses from soil particles. Each method offers different pros and cons, and there are several variables to consider when evaluating the results of each technique. The results will be evaluated statistically, taking into account the nature of the soils from which the extracts were prepared. The most effective and efficient technique will be used further throughout projects done involving soil samples.

Objective 2: Measure viral diversity in order to track changes in the community structure over time and space

In order to properly track the changes in the viral community structure of Lake Matoaka and its watershed, we must determine effective methods for measuring the viral diversity at our sampling sites. For this project, three options are available for measurement: RAPD-PCR1 (randomly amplified polymorphic DNA-PCR); PFGE2 (pulse field gel electrophoresis); and TEM3 (transmission electron microscopy). RAPD-PCR and PFGE use DNA to determine diversity, whereas TEM requires the researcher to visualize viral morphology under an electron microscope. Viral concentrates obtained from samples will be analyzed using these three methods. Once we determine which technique provides the most reliable profile of the sample, we can move on to use the technique in the larger temporal dynamics study.

Objective 3: Study the community structure of viruses in a coastal gradient

Within the limited study of soil viruses to this point, even less work has been done with viruses in sands. Several papers have been published with a focus on the viral community in desert ecosystems4,5, but published work has not been released on viruses in an oceanfront community. The beach offers a unique interface of the terrestrial and marine environments. By looking at samples collected from the Cape Hatteras National Seashore on the Outer Banks of North Carolina, we can take a further step towards learning more about viruses in soil on a whole. Three sites will be sampled from during low tide: at the water-shore interface; between the high and low tide lines; and above the high tide line. Abundance and diversity data will be collected, and inductions will be carried out to check for the presence of temperate bacteriophage. Though the study will be by no means conclusive, the initial step will help us understand soil environments better. This study offers the potential for longer-term, broader-scale work focusing on environmental variables (seasonal factors, tides, etc.).

Timeline

The overall project of temporal dynamics and Lake Matoaka will continue on past the summer, with analyses and processing of data and samples stretching into the late Fall. The studies on efficient extraction and measurement of viral diversity will be completed by the end of the first session, allowing for the effective study of samples collected over the summer. The work on Outer Banks beaches will be completed as work on the Lake Matoaka study allows. Samples will be collected over a weekend in June, then processed shortly after. The preliminary analyses will be completed before the end of the summer. This work will feed into the larger project being carried out by Professor Kurt Williamson, “Temporal Dynamics of Viral Communities in Lake Matoaka and its Watershed.”

References

  1. Winget, D.M., & Wommack, K.E. (2008). Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness. Applied and Environmental Microbiology. 74, 2612-2618.
  2. Steward, G.F. (2001).Fingerprinting viral assemblages by pulsed field gel electrophoresis (PFGE). Methods in Microbiology. 30, 85-103.
  3. Williamson, K.E., Radosevich, M., & Wommack, K.E. (2004). Abundance and diversity of viruses in six delaware soils. Applied and Environmental Microbiology. 71, 3119-3125.
  4. Prigent, M., Leroy, M., Confalonieri, F., Dutertre, M., & DuBow, M.S. (2005). A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the sahara desert. Extremophiles. 9, 289-296.
  5. Prestel, E., Salamitou, S., & DuBow, M.S. (2008). An examination of the bacteriophages and bacteria of the namib desert. The Journal of Microbiology. 46, 364-372.