Abstract
When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.
Highlights
The anaerobic respiratory strategy known as dissimilatory metal reduction likely evolved long before the Earth’s atmosphere became aerobic [1,2], and remains a significant process for geochemical cycling in sediments and subsurface environments [1,3]
Biofilm growth G. sulfurreducens cells attached to poised electrodes (n = 8) with no lag, increased to a current density of .700 mA/cm2, and were all harvested at the same stage of growth (Fig. 1A)
Confocal microscopy of electrodes on which G. sulfurreducens biofilms were grown under similar conditions revealed biofilms of intact cells, based on Live/Dead staining, extending,20 mm from the electrode surface [8,33,34]
Summary
The anaerobic respiratory strategy known as dissimilatory metal reduction likely evolved long before the Earth’s atmosphere became aerobic [1,2], and remains a significant process for geochemical cycling in sediments and subsurface environments [1,3]. A multiheme outer membrane cytochrome (OmcB) known to be regulated in response to environmental conditions [28,29,30,31] was selected as a target for an antibody-based approach for measuring changes in protein abundance within Geobacter anode biofilms. All measurements were performed using biofilms grown on polished anodes, to minimize variability in distance from the electrodes, and multiple high-resolution images were digitally reconstructed to obtain composite images spanning the entire biofilm for each labeling experiment. These data confirmed that direct labeling of resin-embedded Geobacter biofilms can be used to determine protein localization and detect changes in protein abundance throughout a biofilm
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