Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: Aquatic, marine and terrestrial cyanobacteria are important players in global nutrient cycling, are a source of novel biochemical compounds, and are considered a potential source of alternative fuels. Terrestrial cyanobacteria produce significant quantities of material that they excrete beyond their outer membrane creating an extracellular matrix. This material protects them from environmental perturbations, including complete desiccation. We have explored the role of the extracellular matrix of several different cyanobacteria, predominantly through undergraduate research as well as apply similar strategies for the stabilization of mammalian cell lines. Over 12 different undergraduate students have developed microbial and biochemical skill sets through working on these projects. In addition, high school students have worked in our laboratory and helped to create classroom-based projects that serve as microbial educational resources. A concise review of the role of the extracellular matrices in cyanobacteria was prepared for worldwide dissemination in the form of a Book Chapter. In addition, we have helped optimize a stabilization strategy for mammalian cell lines that is currently in use in the public sector. PARTICIPANTS: Undergraduates include: Shane Duncan, Willie Frazier, Jessica Hastie, Benjamin Irvin, Mark Price, Kaila Redifer, Hannah Strawderman, Tara Talaie, Marcy Thompson, Lolly Vilhauer, Amber Villa, Erica Weidman, Nicholas Willoughby. These students received significant training opportunities. Researchers at Virginia Tech include Jody Jervis, Chul Park, Malcolm Potts, W. Keith Ray, and Deborah Wright. One Master's Student (Kevin Horn) completed his degree while working on this project. We also worked extensively with a high school science teacher (Charles Jervis, Auburn High School). TARGET AUDIENCES: Our audience for this work ranged from high school students and teachers to scientists. The development of exercises for use in a high school setting provided context to the learned scientific principle. The Book Chapter on Extracellular Matrices provides a number of hypotheses that should invigorate additional studies on this poorly understood "occupied space." PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The analytical approach taken in characterizing the extracellular matrix of cyanobacteria led to the development of the tools and techniques that permit characterization of materials present outside of cellular membranes. This ability extended the reach of our project beyond its initial goals to include characterizing extracellular matrices of wastewater treatment plants and the development of strategies for the long term storage of mammalian cell lines. The wastewater treatment plant work was the first of its kind as it dealt with actual wastewater treatment samples, not laboratory-generated materials. The mammalian cell stabilization work was initially applied to a commercially-available biosensor presently used by the medical and food industries. Finally, the research efforts led to the training of over 12 different undergraduate students, with many having moved on to graduate, professional, or employed positions.
Publications
- Petrovick MS, Nargi FE, Towle T, Hogan K, Bohane M, Wright DJ, MacRae TH, Potts M, Helm RF. (2010) Improving the long-term storage of a mammalian biosensor cell line via genetic engineering. Biotechnol. Bioeng. 106:474-81.
- Helm RF and Potts MP. Extracellular Matrix. 2011. Chapter 18 in (Whitton BA. ed.) Ecology of Cyanobacteria II, in press. Springer Publishing.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Terrestrial cyanobacteria are a major component of globally distributed microbial communities. Efforts to understand their physiological behavior under laboratory and field conditions are essential for determining their roles in the ecosystems in which they reside. We have described simple protocols to rapidly screen these organisms for the production of "releasable" metabolites. These protocols were developed with the aid of four undergraduate students, who are now trained in the hands-on operation of mass spectrometers. Interactions with high school science teachers and their students have helped create several different classroom-based projects. Five different high school science teaching modules are currently under review by the American Society for Microbiology for addition to their microbial educational resources. PARTICIPANTS: Willie C. Frazier, an undergraduate student at Virginia Tech, participated in this project during the Summer of 2009 through the Multicultural Academic Opportunities Program of Virginia Tech. The program is designed to encourage low-income, first-generation college students and students from groups currently underrepresented in higher education to pursue doctoral study. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Extracellular compounds can have multiple roles important to cellular survival and differentiation. Cyanobacterial extracellular polysaccharides are thought to serve as protectants against desiccation as well as temperature fluctuations, but can also be thought of as a reservoir of low molecular weight molecules. We have shown that growth conditions modulate the metabolites that are produced by these cyanobacteria, suggesting that further work at the chemical and biological levels will provide a better understanding of the dynamic nature of the extracellular space and its role in microbial physiology. Our studies indicate that the chemotypic diversity within cyanobacterial species may be greater than previously anticipated.
Publications
- Park C, Helm RF, Novak JT. 2008. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Water Environ. Res. 80: 2219-2227.
- Jervis J, Templeton S, Potts M, Helm RF. 2009. Analysis of terrestrial cyanobacteria by whole cell MALDI-TOF/TOF mass spectrometry. Environ. Microbiol. Rep., submitted.
- Jervis C, Jervis J, Helm RF. 2009. Using Cyanobacteria in High School Biology and Chemistry Classes. For Publication in the ASM Microbe Library. Chapters submitted and under review: Collection and Ecology; UV Sunscreens, Chromatography and Spectroscopy; Bioinformatics; Cyanobacteria and Extraterrestrial Life (5 total).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We have developed a new high-throughput mass spectrometry tool for evaluating microbial communities. These molecular level studies will lead to a better understanding of the mechanisms of survival for desiccation-tolerant cyanobacteria, thereby permitting a rational approach to designing more efficient packaging systems for labile biologicals Three undergraduate researchers have been mentored in relation to this work. One student evaluated the use of extracellular glycan for stabilizing biomedical products. Another student is developing new strategies for measuring cyanobacterial proteins, while the third is apprenticing in the laboratory as a mass spectrometrist. High school students have worked on our laboratory for science fair projects as well as for training and experimentation with classroom-based endeavors. Field trips from local high schools have provided students the opportunity to see the connection between scientific principles and genuine research. We have developed new collaborative relationships with investigators at the University of Hawaii-Manoa and the University of Massachusetts-Amherst. Our collaborators in Hawaii are providing selected cyanobacterial strains for mass spectral analyses. The researchers in Massachusetts are interested in applying our tools to wastewater treatment facilities in an effort to improve facility practices. One graduate student from our laboratory has completed his Master's thesis in the area of cyanobacterial extracellular matrices. Our laboratory efforts serve as a starting point for introducing scientific principles and practices high schoolers. We continue to interact with teachers and students several counties in Southwest Virginia and have published an on-line tool for the Microbe Library web resource (sponsored by the Amer. Society for Microbiology). PARTICIPANTS: Jody Jervis (Laboratory Technician), Deborah Wright (Laboratory Technician), Kevin Horn (Graduate Student), W. Keith Ray (Research Associate). Collaborators (High School effort): Charles K. Jervis (Auburn High School, Riner VA), James Garst (Salem High School, Salem VA), John Kowalski (Roanoke Valley Governor's School, Roanoke VA); Collaborators (University-based research): Dr. Sean Callahan (University of Hawaii-Manoa), Dr. Ferran Garcia-Pichel (Arizona State University), Dr. Malcolm Potts (Virginia Tech), Dr. Chul Park (University of Massachusetts-Amherst) TARGET AUDIENCES: There are three separate target audiences. Researchers (from molecular ecologists to wastewater treatment engineers) are interested in the mass spectral approach to characterizing microbial communities. The biomedical sector is interested in our efforts to stabilize labile biological products, and high school science teachers are interested in our efforts to bring "real science" to the classroom. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ecological niches dominated by microbes are excellent model systems for understanding how organisms communicate with one another at the molecular level in order to survive. We have developed the mass spectrometry tools necessary to monitor extracellular communication processes, and such tools will provide new knowledge on how communities develop and are maintained. Expansion of our efforts from terrestrial cyanobacteria to wastewater treatment facilities demonstrates the broad applicability of the tools.
Publications
- Park, C., Novak, J.T., Helm, R.F., Ahn, Y.O., Esen A. (2008). Evaluation of the extracellular proteins in full-scale activated sludges. Water Research 42(14):3879-3889.
- Park, C., Helm, R.F. (2008). Application of metaproteomic analysis for studying extracellular polymeric substances (EPS) in activated sludge flocs and their fate in sludge digestion. Water Science Technology, 57(12):2009-2015.
- Horn, K., Jervis, J., Wright, D., Potts, M., and Helm, R. (2008). Roof-Dwelling Desiccation-Tolerant Cyanobacteria (Extremophiles). Visual image web resource. http://www.microbelibrary.org/asmonly/details.aspid=2796&Lang=
- Horn, K. (2008). MS Thesis. The Effect of Nitrates, pH, and Dissolved Inorganic Carbon Concentrations on the Extracellular Polysaccharide of Three Strains of Cyanobacteria Belonging to the Family Nostocaceae
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Activities for this reporting period include evaluation of the extracellular glycan for stabilizing labile proteins (undergraduate research project), using cyanobacterial systems to teach scientific principles to high school chemistry and biology students, and the development of new mass spectrometric techniques for evaluating secondary metabolite and polysaccharide production in cyanobacteria (graduate student PhD research). Key events for disseminating this work include a presentation at the Experimental Biology Meetings held in Washington DC, April 2007, high school science fair projects, and several high school student training and experimentation sessions. This has led to the development of a High School Teacher's Manual and video available that is available at: http://vigen.biochem.vt.edu/outreach.asp. Our development of mass spectral characterization tools for the cyanobacteria have been presented at an international cyanobacterial conference.
PARTICIPANTS: Individuals working on this project include: Jody Jervis (Laboratory Technician), Kevin Horn (Graduate Student), W. Keith Ray (Research Associate), Mark Price (Undergraduate Student), and Tara Wiles (Laboratory Technician). Collaborators include Charles K. Jervis (Auburn High School; Riner, VA) and Malcolm Potts (Professor, Department of Biochemistry, Virginia Tech).
TARGET AUDIENCES: There are two separate target audiences for this work. The biomedical/pharmaceutical sectors are interested in increasing the shelf-life of labile products. The extracellular glycan from cyanobacteria may permit this. Demonstration of long-term storage through publication of peer-reviewed literature will get this message out. The second target audiences are secondary school educators and their students. We have provided numerous opportunities for educators and students to become involved in our work. At present we do not envision increasing the scope of this effort, only fine tuning.
PROJECT MODIFICATIONS: None.
Impacts
The roofs of most residential houses in the United States host a microbial ecosystem that would be classified as extreme. This ecosystem lends itself easily to education and outreach as it is a system the students can observe daily and directly. We utilize this system as a tool to work directly with high school science teachers and students. We are presently utilizing this ecosystem to teach basic scientific principles followed by more in-depth investigations, including the sample isolation and characterization at a site maintained at the participating school. As a result of this program, high school students and teachers will be directly involved in the scientific process of acquiring, interpreting, presenting, and publishing data from these studies. Our work with cyanobacterial isolates also impacts the biomedical field. As the mechanism of survival of these organisms to dehydration and ultraviolet light is based upon the production of an extracellular
polysaccharide, studies aimed at using this material to stabilize labile biological products such as proteins and vaccines may provide a new way to store biologicals without refrigeration. We have found that the extracellular glycan is capable of stabilizing active protein in the dried state under accelerated degradation conditions (heat). The mechanisms of protection and effects of long term (months) storage are currently underway.
Publications
- Jervis CK, Jervis J, Helm RF. 2007. Using cyanobacteria as a tool for integration in high school biology and chemistry classes: a high school-university outreach connection. FASEB JOURNAL 21 (5): A218-A218 April.
- Jervis J, Wright DJ, Horn KJ, Ray WK, Wiles TR, Slaughter SM, Helm RF. 2007. Looking from the outside in: cyanobacterial biofilms, secondary metabolites and cell signaling. 9th Cyanobacterial Workshop, June (Delavan, WI).
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Progress 10/01/05 to 09/30/06
Outputs
Many organisms have the ability to withstand the complete removal of water. Such organisms are of interest for developing approaches to the long term storage of labile biomedical reagents. Preliminary studies have been directed at understanding the organisms found on asphalt shingle roofs, an ecosystem dominated in many parts of the country by the cyanobacterium Gloeocapsa sp. Protocols have been developed for removal of the organism from the shingles for characterization in the laboratory, and we are also in the process of developing several methods to culture the organism in the laboratory. Our initial analyses at the protein and metabolite levels have discovered a phosphate binding protein as well as scytonemin, a UV-protective pigment. Attempts to increase extracellular polysaccharide production in the laboratory via antibiotics has thus far been unsuccessful. In a second path of investigation, Nostoc punctiforme is being used as a model system to determine the
biosynthetic pathways involved in scytonemin biosynthesis. Results to date show that feeding with p-coumaric acid results in an abnormal phenotype that includes deposits of inclusion bodies. This phenomenon is presently under investigation. Finally, as part of the applied aspect of this work, Nostoc commune extracellular polysaccharide is being tested for its ability to stabilize alkaline phosphatase and attenuated viruses during storage.
Impacts
Many microbes naturally cope with the complete loss of water, heat, and intense sunlight through the production of an extracellular matrix (a thick "jacket" or "coat") that surrounds the cells. Work is being directed toward understanding these matrices in order to apply similar strategies for the long term storage of labile biomedical reagents.
Publications
- Wright, D.J., Smith, S.C., Joardar, V., Scherer, S., Jervis, J., Warren, A., Helm, R.F., Potts, M. 2005. UV Irradiation and Desiccation Modulate the Three-dimensional Extracellular Matrix of Nostoc commune (Cyanobacteria). J. Biol. Chem. 280 (2005) 40271-40281.
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