Hydrogen has an extensive potential as a clean and renewable energy source. Photosynthetic, non-sulphur, purple bacteria, Rhodobacter sphaeroides O.U.001 produces molecular hydrogen by nitrogenase enzyme. Nitrogenase enzyme is encoded by nifHDK genes and expression of the structural genes, nifHDK, is controlled by NifA which is encoded by nifA gene. The transcription of nifA is under the control of Ntr system and product of prrA gene. Relationship between the genes that have roles in nitrogenase synthesis should be understood well to increase biological hydrogen production. In this work, expression levels of nitrogenase encoding nifH and control genes nifA, prrA were examined at different physiological conditions. In addition to modifications in expression levels, changes in hydrogen production and growth capacity were also investigated in response to different concentrations of ammonium source, oxygen and different light intensities. In this study, it was found that increasing concentrations of ammonium chloride caused decrease in hydrogen production. Glutamate containing medium had the capacity for higher hydrogen production. The expression levels of nifH and nifA genes decreased with the increase in concentrations of ammonium chloride. There was a negative correlation between the expression levels of prrA gene and its target, nifA gene. Hydrogen production was observed even in aerobic conditions of the same media compositions. It was observed that different culture media had changing growth and hydrogen production capabilities at different light intensities. There was no direct proportion between the expression levels of nifH gene and amount of hydrogen at different light intensities.

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.