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Center for Biofilm Engineering
Abstract:
"Combining in Situ Reverse Transcriptase Polymerase Chain Reaction,
Optical Microscopy and X-ray Photoelectron Spectroscopy to Investigate Mineral
Surface-Associated Microbial Activities"
04-035 A study was undertaken to investigate expression of a gene encoding a c-type
cytochrome in cells of the dissimilatory metal reducing bacterium (DMRB)
Geobacter sulfurreducens during association with poorly crystalline and
crystalline solid-phase Fe(III)-oxides. The gene encoding OmcC (outer membrane
c-type cytochrome) was used as a target for PCR-based molecular detection and
visualization of omcC gene expression by individual cells and aggregates of
cells of G. sulfurreducens associated with ferrihydrite and hematite mineral
particles. Expression of omcC was demonstrated in individual bacterial cells
associated with these Fe-oxide surfaces by in situ RT-PCR (IS-RT PCR) and
epifluorescence microscopy. Epifluorescence microscopy also permitted
visualization of total DAPI-stained cells in the same field of view to assess
the fraction of the cell population expressing omcC. By combining reflected
differential interference contrast (DIC) microscopy and epifluorescence
microscopy, it was possible to determine the spatial relationship between cells
expressing omcC and the mineral surface. Introduction of the fluorescently
labeled lectin concanavalin A revealed extracellular polymeric substances (EPS)
extending between aggregations of bacterial cells and the mineral surface. The
results indicate that EPS mediates an association between cells of G.
sulfurreducens and ferrihydrite particles, but that direct cell contact with the
mineral surface is not required for expression of omcC. XPS analysis revealed
forms of reduced Fe associated with areas of the mineral surface where
EPS-mediated bacterial associations occurred. The results demonstrate that by
combining molecular biology, reflectance microscopy, and XPS, chemical
transformations at a mineral surface can be related to the expression of
specific genes by individual bacterial cells and cell aggregates associated with
the mineral surface. The approach should be useful in establishing involvement
of specific gene products in a wide variety of surface chemical processes.
Magnuson, T.S., A.L. Neal, B.M. Peyton, and G.G. Geesey, "Combining in Situ
Reverse Transcriptase Polymerase Chain Reaction, Optical Microscopy and X-ray
Photoelectron Spectroscopy to Investigate Mineral Surface-Associated Microbial
Activities," Microb. Ecol., 48:578-588 (2004) |
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