Bacterial chemotaxis is the movement of bacteria towards or away from chemical stimuli in the surrounding media. Bacteria respond to chemotactic signals through chemoreceptors which bind specific ligands and transduce signals through a modified two-component system. Typical chemoreceptors bind a ligand in the periplasm and signal across the inner membrane to the cytoplasmic chemosensory array through the inner membrane. Bacterial chemoreceptors must integrate multiple signals within an array of different receptor homologues to a single output. Chemoreceptors act cooperatively to allow a rapid signal spread across the array and large signal gain. Chemoreceptors adapt to a signal by chemical modification of their cytoplasmic domains in order respond across a wide range of effector concentrations. How bacterial chemoreceptors transduce signals through the inner membrane, integrate multiple effector responses, signal cooperatively and adapt to result in a single output signal is not currently fully known. In Rhodobacter sphaeroides, additional complexity arises from the presence of multiple homologues of various chemotactic components, notably the array scaffold protein CheW. Decoding this signalling mechanism and heterogeneity involved in this system is important in decoding the action of a biological system, with implications for biotechnology and synthetic biology. This study used the two model systems Escherichia coli and R. sphaeroides to analyse the mechanism of signalling through bacterial chemoreceptors. Rational design of activity-shifting chemoreceptor mutations was undertaken and these variants were analysed in phenotypic and fluorescence localisation studies. Molecular-dynamics simulations showed an increase in flexibility of chemoreceptors corresponds to a decrease in kinase output activity, which was determined by the computational tracking of bacteria free-swimming in media. Fluorescence recovery after photobleaching was used to show that this increase in flexibility results in a decrease in binding of receptors to their array scaffold proteins. A two-hybrid screen also suggested that inter-receptor affinity is also likely to decrease. These results show that signalling through chemoreceptors is likely through a mechanism involving the selective flexibility of chemoreceptor cytoplasmic domains. Analysis of R. sphaeroides chemoreceptors and CheW scaffold proteins in E. coli showed that it should be possible to design, from the bottom-up, a functional bacterial chemotaxis system in order to analyse individual protein specificity. Expression of R. sphaeroides MCPs in this E. coli system show the reconstitution of a chemotactic array, but not one capable of signalling specifically to proposed attractants. Results gained from this system suggest the R. sphaeroides CheW proteins are not homologous and their differential binding affinities may allow array activity 'fine-tuning'.

One of the most important parameters affecting hydrogen production in photofermentation process is the type of carbon and nitrogen sources. For this reason in this research, the effect of different nitrogen sources (5mM ammonium chloride and 2mM glutamate) and acetate concentrations (40

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.

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.

photosynthetic bacteria is attractive field as production is fueled by solar energy. Hydrogen production potential of two photosynthetic bacteria R.capsulatus (DSM1710 wild type and R.capsulatus YO3 Hup- uptake hydrogenase deleted mutant strain) were examined in agar immobilized systems. In the present work agar and glutamate concentrations were optimized for immobilization of bacteria while feeding bacteria with 40/2-4 mM acetate/ glutamate. Immobilized bacteria produced hydrogen for 420-1428 hours covering 5-7 rounds. Optimizing of acetate concentration indicated that 60 mM produced the highest observed yield around 90-95%. Results shown that 2.5 mg dry cell weight/mL is the optimum cell concentration for wild type strain while 5 mg dry cell weight/mL was optimum for YO3 strain. Using either glycerol or sodium dithionite caused decrease in hydrogen production capacity of immobilized bacteria. It was observed that agar provided protection against inhibition effect of ammonium. Co- v immobilization of bacteria with packed cells of H. salinarium increased total hydrogen production capacity by about 1.14-1.41 folds. Hydrogen production by immobilized bacteria in panel photobioreactor was achieved by a novel system which allowed long term hydrogen production. Immobilized R. capsulatus DSM 1710 in panel reactor worked for about 67-82 days covering 4-5 rounds while immobilized R. capsulatus YO3 worked for 69-72 days covering seven rounds.

Rhodobacter capsulatus DSM1710 is a purple non-sulfur bacterium capable of hydrogen production via photofermentation. Biohydrogen is a clean and renewable way of hydrogen production, which can be achieved by PNS bacteria in outdoor large scale photobioreactors using sun light. In outdoor conditions bacteria can be exposed to heat and cold stress. In this study in order to understand the effects of heat and cold stress on photofermentative hydrogen production and gene expression profile of R.capsulatus on acetate as the carbon source, microarray analysis was carried out. Since there is no commercially available microarray chip for R.capsulatus, an Affymetrix GeneChip&reg / was designed and it was manufactured by Affymetrix.The experiments were conducted at 30

Hydrogen, which is a clean energy source, is one of the alternatives for fossil fuels. Biological hydrogen production is one of the hydrogen production methods. Rhodobacter capsulatus is a photosynthetic bacterium that produces hydrogen via photofermentation. R. capsulatus can also synthesize some valuable by-products such as Poly-beta- hydroxy butyric acid (PHB), which is a biodegradable bioplastic. In a two stage biohydrogen production system, which is combination of dark fermentation and photofermentation, dark fermentor effluents are used for photofermentation by R.capsulatus. Dark fermentor effluents usually contain high amount of acetate. High amount of acetate may decrease the efficiency of hydrogen production by causing high amount of PHB production. Therefore, it is significant to determine optimum acetate concentration for photofermentation. In this study, the effects of acetate concentration on hydrogen and PHB production by R.capsulatus were investigated by growing bacteria at various acetate concentrations (10 mM-65 mM). In addition, gene expression analysis was performed to investigate the effects of acetate at transcriptional level. For this purpose, expression levels of the genes that encode nitrogenase which is the enzyme that catalyzes hydrogen production and PHB synthase, which is the key enzyme of the PHB synthesis pathway, are examined. Optimum acetate concentration for photofermentation with high hydrogen yield and low PHB amount was determined to be in the range 25 mM-50 mM. nifD expression was found to be high at optimum acetate concentrations and phaC expression was found to be the highest at 65 mM.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 184-198).

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.

Facultative phototrophic bacterium Rhodobacter capsulatus has two c-type electron carrier cytochromes (cyt) / the soluble cyt c2 and the membrane-attached cyt cy, that act as electron carriers during respiratory and photosynthetic growth of this species. Previously, a soluble form of cyt cy was constructed by fusing genetically the signal sequence of cyt c2 to the cyt c domain of cyt cy. The obtained novel soluble cyt cy (cyt S-cy) was unable to support photosynthetic growth of R. capsulatus but yielded photosynthetically functional (Ps+) revertants frequently. In the first part of this study, photosynthetic electron transfer properties of some of Ps+ revertants of cyt S-cy were analyzed by biochemical and biophysical methods and compared with the cyt cy and cyt c2. Reduction-oxidation titration of membrane supernatants showed that the redox midpoint potential of cyt S-cy was +338 mV which is similar to midpoint potentials of cyt cy or the cyt c2. However, light-activated, time resolved spectroscopy revealed that reaction center mediated oxidation kinetics of cyt S-cy exhibited only a slow phase, unlike cyt c2 which has both fast and slow phases. It therefore appeared that during electron transfer cyt S-cy does not interact with the reaction centre as tightly as cyt c2. These findings imply that attaching electron carrier cyts to the membrane allowed them to weaken their interactions with their partners, while restricting their spatial diffusion, so that they accomplish rapid multiple turnovers. In the second part of this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes, cyt cbb3 deleted and Qox deleted strains were compared with the wild type. Under photoheterotrophic growth conditions with limiting nitrogen source, the excess reducing equivalents generated by organic acid oxidation are consumed to reduce protons into hydrogen by the activity of nitrogenase in R. capsulatus. The results indicated that the hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3-5 folds, and the hydrogen production rate of the cyt cbb3- mutant increased significantly. Moreover in this study, the hydrogen production efficiency of different R. capsulatus strains was increased by the chromosomal inactivation of uptake hydrogenase genes and enzymatic activity of uptake hydrogenase of R. capsulatus strains were determined.