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)

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>

Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated. Nostoc punctiforme PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by hupSL. The transcription of hupSL was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in N. punctiforme, the hypFCDEAB operon. These genes were found to be situated upstream of hupSL in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon. The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of N. punctiforme was constructed, where hupL was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the hupL- mutant and in wild type N. punctiforme was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup- mutant of the related strain Nostoc PCC 7120 were also studied. This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.