CHARACTERIZATION OF OUTER MEMBRANE PROTEINS INVOLVED IN IRON REDUCTION AND BIOFILM FORMATION IN GEOBACTER SULFURREDUCENS

Open Access
Author:
Stephen, Camille Simone
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 14, 2011
Committee Members:
  • Ming Tien, Dissertation Advisor
  • Ming Tien, Committee Chair
  • Susan Louise Brantley, Committee Chair
  • Jean Elnora Brenchley, Committee Member
  • Squire J Booker, Committee Member
  • James Gregory Ferry, Committee Member
Keywords:
  • Geobacter sulfurreducens
  • biofilm
  • iron reduction
  • electrode
Abstract:
Polyclonal antibodies raised against the outer membrane cytochrome OmcB and the cytoplasmic protein acetate kinase were used to localize these proteins in a multi-layer Geobacter biofilm grown on a poised electrode. OmcB is a cytochrome that is important for both the reduction of soluble and insoluble iron(III) (Leang et al., 2003). Acetate kinase is involved in the metabolism of acetate (Galushko and Schink, 2000). Acetate served as the electron donor and carbon source for Geobacter cells in all experiments. Immunolocalization of these proteins in the biofilm was visualized with transmission electron microscopy. OmcB labeling of the biofilm revealed that OmcB was more abundant at the outer membrane of cells in the outer layers of the biofilm compared to cells closer to the electrode surface. In contrast, acetate kinase was expressed evenly throughout the biofilm. The difference in the abundance of OmcB in the biofilm is consistent with electron acceptor limitation for cells in the outer layers of the biofilm. Differences in protein expression between current-producing biofilm cells and fumarate-grown planktonic cells were investigated using iTRAQ-MS analysis. Some of the proteins that were up regulated in the biofilm cells relative to the planktonic cells are involved in cell division, protein folding, sensing environmental changes or stimuli, signal transduction, DNA synthesis and oxidative stress response. Some of the proteins that were down regulated in the biofilm cells are proteins involved in fumarate metabolism, proteolysis and oxidative stress response. One outer membrane cytochrome (GSU2811) was up regulated in the biofilm cells. Other outer membrane cytochromes such as OmcB, OmcE, OmcS, which play important roles in the iron reduction pathway (Leang et al., 2003; Mehta et al., 2005) were not identified in the iTRAQ study. OmcZ, which is involved in electron transfer to electrodes (Nevin et al., 2009), was identified but not labeled by the iTRAQ reagents and PilA, which is the pilin subunit for the proposed conductive nanowires (Reguera et al., 2005), was also not identified in the iTRAQ study. To determine the expression of these proteins in the current-producing biofilm cells compared to the fumarate-grown planktonic cells, antibodies produced against the purified protein or peptides were used in Western blotting. Both OmcB and OmcS showed a slight difference in expression with slightly less OmcB and slightly more OmcS in the biofilm cells relative to the planktonic cells. Acetate kinase protein AckA-1 was also slightly more intense in the biofilm cells compared to the planktonic cells. More significant differences in expression were seen for OmcZ, PilT-4 and PilA. The 30 kDa OmcZ was more abundant in the biofilm cells compared to the planktonic cells and some of the 50 kDa OmcZ was detected in the planktonic cells, but not in the biofilm cells, indicating incomplete processing of the 50 kDa OmcZ protein by the subtilisin protease in the planktonic cells. Also, PilT-4 was more abundant in the biofilm cells compared to the planktonic cells. PilA however showed no trend in its abundance between biofilm cells and planktonic cells. A difference in the abundance of the outer membrane cytochrome OmcB in the current-producing biofilm based on the cells’ proximity to the anode was demonstrated in this study. In addition, differences in expression for various outer membrane cytochromes and pili proteins were also evaluated. These findings show that G. sulfurreducens alters the expression of these outer membrane proteins in response to biofilm growth and its limitations.