Geobacter sulfurreducens is a dissimilatory iron-reducing bacterium that is able to utilize insoluble external electron acceptors such as Fe(III) oxides, radionuclides, and the anode of a microbial electrochemical cell (MEC). To accomplish this process it uses direct contact mechanisms involving a host of c-type cytochromes. It also produces microbial nanowires that are necessary for efficient growth with all of the aforementioned insoluble electron acceptors. The question is then: Are these nanowires transferring electrons to the acceptors, or are they merely a scaffold for electron transporting cytochromes. Thus, I investigated the contribution of electroactive pili to electron transfer (ET) to external electron acceptors as well as the mechanism of electron transfer in the pili. In chapter 2 I demonstrate the necessity of nanowires for electron transfer through an anode biofilm by generating a mutant that displays only pili defects. Deleting gene encoding the pilus motor, PilB, produces a pili-deficient mutant with wild-type cytochrome expression that formed anode biofilms as thick (ca. 10 μm) as the wild type yet with reduced electroactivity. Furthermore, the growth and electroactivity of thicker biofilms required the expression and conductivity of the pili. The results support a model in which the conductive pili form a nanopower grid that permeates the biofilms to wire the cells in all biofilm strata to the underlying electrode. The pili operate coordinately with cytochromes in the lower strata until the biofilm reaches a threshold thickness where the pili are required as electronic conduits and thus without this function the biofilm is unable to continue to grow. In chapter 3 I investigate the mechanism of pilus conductivity by generating amino acid replacements of residues predicted to be involved in ET in the pilus. Pili isolated from a Y27A mutant strain displayed a decreased ability to transfer electrons along the length of the pilus. This mutant, however, displayed no defects in biological external electron transfer assays. Electron transfer rates explained this phenotype, as the mutated pili were still able to transfer electrons faster than the acetate respiration rates. Two additional tyrosine residues, Y32 and Y57 were essential for MEC electroactivity, as were the negatively charged amino acids D53 and D54. Replacement of the three tyrosine residues with the aromatic amino acid phenylalanine, however, resulted in a strain with no reduction in electroactivity. Thus, the presence of these aromatic and charged amino acids is required for optimal charge transfer in the G. sulfurreducens pilus. In Chapter 4 I develop a system to determine the role of intramolecular ET of the pilin monomer. To this end, I collaborated with Dr. Castro-Forero of the Worden Lab to generate a method for the in vitro production of soluble pilin monomers. These recombinant pilins were able to form conductive filaments in vitro and thus retain their ET capabilities. I then modified these recombinant pilins to attach to a gold electrode and used these self-assembled monolayers to study the role of intramolecular electron transfer. I generated several point mutations in the codons for aromatic and charged residues predicted to be involved in ET.
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