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.
Bibliographical noteFunding Information:
Thank you to Gilbert Ahlstrand at the University of Minnesota for embedding biofilms for TEM, also Dr. Gang Ning and Missy Hazen of the Penn State Microscopy and Cytometry Facility (University Park, PA) for assistance with sample preparation for TEM. Thank you to Dr. Scott Geib and Dr. Ruth Nissly for suggestions on data analysis and Dr. Bruce Logan and Karl Shellenberger for G. sulfurreducens DNA. Some biofilms used for preliminary antibody testing were grown by Dr. Douglas Call. D. R. B. was supported by the Office of Science (BER), U.S. Department of Energy, DE-SC0006868.
© 2014 Stephen et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.