The Geobacteraceae are a family of deltaproteobacterial anaerobes known to play
important roles in environmental Fe(III)-reduction, subsurface petroleum
bioremiediation, bioprecipitation of heavy metals, and reduction of anodes in microbial
fuel cells. Electron transfer by Geobacteraceae requires formation of a multicellular
biofilm, as cells must attach to surfaces or insoluble particles to bring redox proteins in
contact with their electron acceptor, and growth of daughter cells requires cell-cell
contact to facilitate longer-range electron transfer. The primary aim of this thesis was to
develop new tools for three-dimensional visualization of gene expression within these
biofilms. To detect differential expression in a developing biofilm, the genetically
tractable representative Geobacter sulfurreducens was utilized as a model. Promoters
from two c-type cytochromes proven to be important in electron transfer were inserted
upstream of the fluorescent protein mCherry, or a derivative of mCherry containing an
additional peptide sequence (LVA) to accelerate protein degradation and act as a signal
of more recent expression. Control plasmids containing no mCherry, or with the
fluorescent protein under control of a constitutive taclac promoter were also transformed
into G. sulfurreducens. The plasmid backbone used for expression was found to affect
growth of cells as biofilms, and a new mobilizable plasmid was developed for use in
these experiments. Cells had to be exposed to oxygen for four hours to achieve
maximum fluorescence, indicating that future real-time studies with these proteins reliant
on oxygen leakage into biofilms would not be reliable. In addition, as G. sulfurreducens
was found to possess significant background fluorescence when grown as a biofilm in the
same emission range as mCherry, future studies should investigate reporters that operate
at different wavelengths. After correcting for these effects, it was demonstrated the
promoter for omcZ was expressed fairly uniformly throughout the biofilm, with localized
pockets of high expression. The promoter for omcS, was expressed at significantly higher levels towards the top of the biofilm when biofilms were at maximum thickness (>20
μm). These results demonstrate that promoter-reporter fusions can be used for
visualization of cytochrome expression in G. sulfurreducens biofilms, and highlights
numerous issues related to plasmid choice, protein oxidation, and background
fluorescence which could create artifacts and confound results.
University of Minnesota M.S. thesis. December 2010. Major: Microbial Engineering. Advisor: Dr. Daniel R. Bond PhD. 1 computer file (PDF); vi, 63 pages.
Remarcik, Clint Michael.
Visualizing gene expression in geobacter sulfurreducens biofilms on graphite electrodes.
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