Geobacter sulfurreducens possesses type IV pili that are considered to be conductive nanowires and a crucial structural element in biofilm formation, enabling electron transfer to insoluble metal oxides in anaerobic sediments and to graphite anodes in microbial fuel cells. The molecular mechanism by which electrons are transferred through the nanowires to the electron acceptor is not fully understood. Prior to the work described in this thesis, the gene (pilA) encoding the structural pilus subunit had been identified, but little was known about the functional translation start codon, the length of the mature secreted protein, or what renders the pili conductive. Using mass spectrometry, I found that a tyrosine residue (Y32) near the carboxyl terminus of the mature PilA protein is posttranslationally modified by attachment of glycerophosphate. I studied the significance of Y32 for biofilm formation on various surfaces and for growth of G. sulfurreducens with insoluble electron acceptors. A mutant in which Y32 was replaced by phenylalanine lacked the glycerophosphate; biofilm formation on graphite surfaces was severely diminished and current production in microbial fuel cells was initiated only after a long lag phase. Moreover, cells with Y32F mutation in the pilA gene exhibited growth deficiency when Fe(III) oxide was the sole electron acceptor. My data confirm the role of G. sulfurreducens pili in biofilm formation and electron transfer to Fe(III) oxide and identify an amino acid in the PilA protein that is essential for these two processes. I also confirmed the existence of two functional translation start codons for the pilA gene and identified two isoforms (short and long) of the PilA preprotein by series of genetic complementation experiments. The short PilA isoform is found predominantly in an intracellular fraction, and seems to stabilize the long isoform and influence the secretion of several outer surface c-type cytochromes. The long PilA isoform, on the other hand, is required for secretion of PilA to the outer surface of the cell, a process that requires co-expression of pilA and the nine genes on its 3’ side. The long isoform is essential for biofilm formation on various surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxide. This study provides new insight concerning the function and biogenesis of Geobacter type IV PilA, as well as a foundation for further research that will be conducted on microbial nanowires.

Pseudomonas aeruginosa uses type IV pili (T4P) to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically-distinct alleles of pi/A in P. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (Kus et al., Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PA01 pi/A mutant. Expression of group I or IV pi/A genes restored twitching motility to the same extent as the PA01 group II pilin. In contrast, complementation with group Ill or group V pi/A genes resulted in poor twitching that increased significantly when the cognate tfp Y or tfpZ accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili, and not to alterations in total pilin pools. Expression of the pilin genes, with or without accessory genes, in a PA01 pi/A-pi/T double mutant background resulted in expression of large amounts of surface pili, suggesting that the accessory proteins function to modulate pilin retraction dynamics. Reduction of twitching motility and surface piliation was also observed a tfpYknockout mutant of group Ill strain PA14, confirming that the accessory proteins enhance pilus assembly on the cell surface. The accessory proteins are specific for their cognate pilins; a PilAv-TfpY chimera produced few surface pili, resembling the phenotype of PA01 complemented with pi!Av alone. The linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins antagonize pilus retraction, increasing pilus expression on the cell surface and thereby enhancing function. / Thesis / Master of Science (MSc)