Yutao Fu

Date of Award

12-2002

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Daniel L. Distel

Second Committee Member

Katherine J. Boettcher

Third Committee Member

John T. Singer,

Abstract

Protistan bacterivory can influence the size distribution, cell structure and composition of natural bacterial communities and is of significant concern for design of bioremediation efforts, yet adequate methods for observation and modeling are lacking. In this investigation, fluorescent protein expression and flow cytometry were used to study protistan grazing on genetically modified strains of several bacterial species that have been considered for use in bioremediation. Broad-host-range plasmids were constructed and used to introduce genes encoding GFP (green fluorescent protein) or RFP (red fluorescent protein) to prey species. A heterotrophic flagellate Paraphysomonas imperforata (Hflag) served as a model predator. Predator-prey interactions were observed and quantified using particle counts and individual optical signals recorded by FACScan flow cytometry. Result files were parsed by Windows Multiple-Document flow cytometry Interface (WinMDI v2.8) and analyzed by GR, a Per1 (Practical Extraction and Report Language) program described herein. Grazing preference of Hflag was influenced by prey type, size and predator culturing conditions. Hflag showed strong preference for bacterial cells over algal cells of a similar size, as well as for bacterial cells of different dimensions. However, size preferences were observed among cells within individual bacterial prey species. Significant preference was also observed for cells labeled by GFP and for unstained cells as compared to cells stained by a traditional DTAF-staining (5- (4,6-Dichloro-Triazin-2-y1)-Amino Fluorescein hydrochloride) method. No difference was observed between cells labeled by GFP and unstained cells. These results show that the methods described here provide a viable approach to observing protistan bacterivory that is superior in some respects to currently used methods.

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