Rhodobacter sphaeroides O.U.001 is a purple non-sulphur bacterium producing hydrogen under photoheterotrophic, nitrogen limited conditions. Hydrogen is produced by Mo-nitrogenase but substantial amount of H2 is reoxidized by a membrane bound uptake hydrogenase. In this study, hydrogen production and the expression of structural nitrogenase genes were investigated by varying molybdenum and iron ion concentrations. These two elements are found in the structure of Mo-nitrogenase and they are important for functioning of the enzyme. The results showed that hydrogen production and nifD gene expression increased upon increase in molybdenum concentration. Increasing iron concentration had also positive effect on hydrogen production and nifK gene expression. To improve the hydrogen producing capacity of R. sphaeroides O.U.001, hupSL genes encoding uptake hydrogenase were disrupted in two different methods. In the first method, hup genes were disrupted by gentamicin resistance gene insertion. In the second method, part of the hup gene was deleted without using antibiotic resistance gene. The wild type and the hup- mutant cells showed similar growth patterns but substantially more hydrogen was produced by the mutant cells. The genes coding for hox1 hydrogenase of Thiocapsa roseopersicina was aimed to be expressed in R. sphaeroides O.U.001 to produce H2 under nitrogenase repressed and mixotrophic conditions. The hox1 hydrogenase genes of T. roseopersicina were cloned and transferred to R. sphaeroides. Although the cloning was successful, the expression of hydrogenase was not achieved by using either the native promoter of hox1 hydrogenase or the crtD promoter of T. roseopersicina.

All the eighteen Frankia strains isolated from ten different actinorhizal host plants showed uptake hydrogenase activity. The activity of this enzyme is further increased by addition of nickel. Nickel also enhanced the degree of hydrogenase transfer into the membranes of Frankia, indicating the role of this metal in the processing of this enzyme. The uptake hydrogenase of Frankia is most probably a Ni-Fe hydrogenase. Genome characterization revealed the presence of two hydrogenase genes (syntons) in Frankia, which are distinctively separated in all the three available Frankia genomes. Both hydrogenase syntons are also commonly found in other Frankia strains. The structural, regulatory and accessory genes of both hydrogenase synton #1 and #2 are arranged closely together, but in a clearly contrasting organization. Hydrogenase synton #1 and #2 of Frankia are phylogenetically divergent and that hydrogenase synton #1 is probably ancestral among the actinobacteria. Hydrogenase synton #1 (or synton #2) of Frankia sp. CcI3 and F. alni ACN14a are similar in gene arrangement, content and orientation, while the syntons are both reduced and rearranged in Frankia sp. EANpec. The hydrogenases of Frankia sp. CcI3 and F. alni ACN14a are phylogenetically grouped together but never with the Frankia sp. EAN1pec, which is more closely related to the non-Frankia bacteria than Frankia itself. The tree topology is indicative of a probable gene transfer to or from Frankia that occurred before the emergence of Frankia. All of the available evidence points to hydrogenase gene duplication having occurred long before development of the three Frankia lineages. The uptake hydrogenase synton #1 of Frankia is more expressed under free-living conditions whereas hydrogenases synton #2 is mainly involved in symbiotic interactions. The uptake hydrogenase of Frankia can also be manipulated to play a larger role in increasing the efficiency of nitrogen fixation in the root nodules of the host plants, there by minimizing the need for environmentally unfriendly and costly fertilizers. The hydrogen-evolving hydrogenase activity was recorded in only four Frankia strains: F. alni UGL011101, UGL140102, Frankia sp. CcI3 and R43. After addition of 15mM Nicl2, activity was also detected in F. alni UGL011103, Frankia sp. UGL020602, UGL020603 and 013105. Nickel also increased the activity of hydrogen-evolving hydrogenases in Frankia, indicating that Frankia may have different types of hydrogen-evolving hydrogenases, or that the hydrogen-evolving hydrogenases may at least be regulated differently in different Frankia strains. The fact that Frankia can produce hydrogen is reported only recently. The knowledge of the molecular biology of Frankia hydrogenase is, therefore, of a paramount importance to optimize the system in favor of hydrogen production. Frankia is an attractive candidate in search for an organism efficient in biological hydrogen production since it can produce a considerable amount of hydrogen.

TRANSCRIPTIONAL ANALYSIS OF HYDROGENASE GENES IN RHODOBACTER SPHAEROIDES O.U.001 In photosynthetic non-sulphur bacteria, hydrogen production is catalyzed by nitrogenases and hydrogenases. Hydrogenases are metalloenzymes that are basically classified into: the Fe hydrogenases, the Ni-Fe hydrogenases and metal-free hydrogenases. Two distinct Ni-Fe hydrogenases are described as uptake hydrogenases and bidirectional hydrogenases. The uptake hydrogenases are membrane bound dimeric enzymes consisting of small (hupS) and large (hupL) subunits, and are involved in uptake and the recycling of hydrogen, providing energy for nitrogen fixation and other metabolic processes. In this study the presence of the uptake hydrogenase genes was shown in Rhodobacter sphaeroides O.U.001 strain for the first time and hupS gene sequence was determined. The sequence shows 93% of homology with the uptake hydrogenase hupS of R.sphaeroides R.V. There was no significant change in growth of the bacteria at different concentrations of metal ions (nickel, molybdenum and iron in growth media). The effect of metal ions on hydrogen production of the organism was also studied. The maximum hydrogen gas production was achieved in 8.4&micro / M of nickel and 0.1 mM of iron containing media. The expression of uptake hydrogenase genes were examined by RT-PCR. Increasing the concentration of Ni++ up to 8.4&micro / M increased the expression of uptake hydrogenase genes (hupS). At varied concentrations of Fe-citrate (0.01 mM-0.1 mM) expression of hupS was not detected until hydrogen production stopped. These results will be significant for the improvement strategies of Rhodobacter sphaeroides O.U.001 to increase hydrogen production efficiency. In order to examine the presence of hupL genes, different primers were designed. However, the products could not be observed by PCR.

<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.