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'.

The main objective of this study is exploring the parameters affecting photobiological hydrogen production and developing anaerobic photobioreactor for efficient photofermentative hydrogen production from organic acids in outdoor conditions. Rhodobacter capsulatus and Rhodobacter sphaeroides strains were used as microorganisms. EU project &ldquo / Hyvolution&rdquo / targets to combine thermophilic fermentation with photofermentation for the conversion of biomass to hydrogen. In this study, the effluent obtained by dark fermentation of Miscanthus hydrolysate by T. neapolitana was fed to photobioreactor for photofermentation by R. capsulatus. Hydrogen yield was 1.4 L/Lculture showing that the integration of dark and photofermentation is possible. Innovative elements were introduced to the photobioreactor design such as removal of argon flushing. An online gas monitoring system was developed which became a commercial product. It was found that the light intensity should be at least 270 W/m2 on the bioreactor surface for the highest hydrogen productivity and the hydrogen production decreased by 43 % if infrared light was not provided to the bioreactor. Scale-up of photofermentation process to 25L was achieved yielding 27L hydrogen in 11 days by R. capsulatus on acetate/lactate/glutamate (40/7.5/2 mM) medium. The outdoor application of the system was made. Shading and water spraying were adapted as cooling methods for controlling the temperature of the outdoor bioreactor. It was found that uptake hydrogenase deleted mutant of R. capsulatus show better hydrogen productivity (0.52 mg/L.h) compared to the wild type parent (0.27 mg/L.h) in outdoor conditions. It was also shown that the hydrogen production depended on the sunlight intensity received.

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.

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.

Thesis (Doctoral)--Technische Universität Carolo-Wilhelmina zu Braunschweig, 1982.

Universiẗat, Diss., 2003--München.

Bacteria swim towards improving conditions by controlling flagellar activity via signals (CheY) sent from chemosensory protein clusters, which respond to changing stimuli. The best studied chemotactic bacterium, E. coli, has one transmembrane chemosensory protein cluster controlling flagellar behaviour. R. sphaeroides has two clusters, one transmembrane and one cytoplasmic. The roles of the two clusters in regulating swimming and chemosensory behaviour are explored here. Newly-developed software was used to measure the effect of deleting or mutating each chemotaxis protein on unstimulated swimming and on the chemosensory response to dynamic change. New behaviours were identified by using much larger sample sizes than previous studies. R. sphaeroides chemotaxis mutants were classified as (i) stoppy unresponsive; (ii) smooth unresponsive or (iii) stoppy inhibited compared to wildtype swimming and chemosensory behaviour. The data showed that the ability to stop during free-swimming is not necessarily connected to the ability to respond to a chemotaxis challenge. The data suggested a new model of connectivity between the two chemosensory pathways. CheY₃ and CheY₄ are phosphorylated by the transmembrane polar cluster in response to external chemoeffector concentrations. CheY₆-P produced by the cytoplasmic cluster is a requirement for chemotaxis, whether or not the polar cluster is able to produce CheY₆-P. CheY₆-P stops the motor, whereas CheY_3,4-P allow smooth swimming. When chemoeffector levels fall, the signals through CheY_3,4 fall, allowing CheY₆-P to bind and stop the motor. As the polar cluster adapts to the fall by the action of the adaptation proteins CheB₁ and CheR₂, the concentration of CheY_3,4-P increases again, to compete with CheY₆-P and allow periods of smooth swimming. Under aerobic conditions, the cytoplasmic cluster controls the basal stopping frequency and does not appear to respond to external chemoeffector changes. The role of the adaptation proteins in resetting the signalling state in R. sphaeroides is unclear, particularly the roles of the proteins associated with the cytoplasmic cluster, CheB₂ and CheR₃. Tandem mass spectrometry was used to identify glutamate and glutamine (EQ) sites on the cytoplasmic R. sphaeroides chemoreceptor TlpT that are deamidated and methylated by the R. sphaeroides adaptation homologues. In E. coli, adaptation sites are usually EQ/EQ pairs. However the sites reported in TlpT vary at the first residue in the pair. Mutation of the putative EQ adaptation sites caused changes in adaptation, suggesting that CheY₆-P levels are controlled and reset by CheB₂ and CheR₃ controlling the adaptation state of TlpT.

Durch die vorliegende Arbeit wurde gezeigt, dass Rhodobacter sphaeroides das Potenzial besitzt, umweltverträglich photoheterotroph Wasserstoff als alternativer, erneuerbarer Energieträger zu erzeugen. Aus genomischen und transkriptomischen Erkenntnissen konnten Rückschlüsse auf Ansatzpunkte für weitere Optimierungen getroffen werden. Durch ein neues Minimalmedium, welches zukünftig sogar einen Beitrag zur Abfallbeseitigung leisten kann, wurde ein wichtiger Schritt hinsichtlich der industriellen Anwendbarkeit von R. sphaeroides für die biologische Wasserstoffproduktion gemacht.:Danksagung Datenverfügbarkeit Inhaltsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis 1. Einleitung 1.1 Wasserstoff 1.1.1 Wasserstoff als Energieträger 1.1.2 Herstellung von Wasserstoff 1.1.2.1 Konventionelle Wasserstoffproduktion 1.1.2.2 Biologische Wasserstoffproduktion 1.1.2.3 Biologische Wasserstoffproduktion aus Abfällen 1.2 Photosynthetische Bakterien 1.2.1 Rhodobacter sphaeroides im Kontext der biologischen Wasserstoffproduktion 1.2.2 An der Wasserstoffproduktion beteiligte Enzyme 1.3 Third Generation-Sequencing Technologien 2. Zielstellung 3. Material 3.1 Chemikalien 3.2 Medien und Pufferlösungen 3.2.1 Van Niel´s Yeast Medium 3.2.2 Medium nach Krujatz et al. (2014) 3.2.3 RÄ-Medium nach Mougiakos et al. (2019) 3.2.4 PY (Peptone Yeast) Agarmedium 3.2.5 2x YT Medium 3.2.6 LB Medium 3.2.7 GYCC Medium 3.2.8 SOB Medium 3.2.9 SOC Medium 3.2.10 Pufferlösungen 3.3 Mikroorganismen 3.4 Molekularbiologische Reagenzien und Primer 3.5 Plasmide 3.5.1 pCas9 3.5.2 pRKPOL2 3.5.3 pSUPPOL2Sca 3.5.4 pBBRBB-Ppuf843-1200-DsRed 3.5.5 pBBR_cas9_NT 3.6 Geräte 4. Methoden 4.1 Rhodobacter sphaeroides Dauerkultur in Van Niel´s Yeast Medium 112 (ohne Wasserstoffproduktion) 4.2 Rhodobacter sphaeroides Batch-Kultivierung 4.2.1 Kultivierung in Medium nach Krujatz et al. (2014); Vollmedium mit Wasserstoffproduktion 4.2.2 Kultivierung in Fruchtsaftmedium 4.3 Rhodobacter sphaeroides Kultivierung mit kontinuierlicher Aufzeichnung von Temperatur, pH, optischer Dichte, Wasserstoffproduktion und Gasanalyse 4.4 Zellernte 4.5 Nukleinsäureextraktion mit dem MasterPureTM Complete RNA and DNA Purification Kit 4.6 DNase-Abbau 4.7 RNase-Abbau 4.8 Qualitätskontrolle der RNA und DNA mit dem Agilent 2100 Bioanalyzer 4.9 Reverse Transkription und Probenaufreinigung 4.10 qRT-Polymerasekettenreaktion 4.11 Etablierung der CRISPR-Cas9- Methodik bei Rhodobacter sphaeroides – Gen-Knockout der Hydrogenase Untereinheit hupL mit CRISPR-Cas9 4.11.1 Anzucht der Escherichia coli Stämme mit und ohne Plasmid 4.11.2 Plasmid Extraktion mit GeneJET Plasmid Miniprep Kit (#K0502, Thermo Scientific) 4.11.3 Restriktionsverdau zur Vektorlinearisierung 4.11.4 Design der guideRNA 4.11.5 Phosphorylierung der guideRNA 4.11.6 Ligation der guideRNA in pCas9 4.11.7 Transformation pCas9_hupL1/hupL2 in Escherichia coli JM109 durch chemische Kompetenz 4.11.8 Colony-PCR zum Insertnachweis hupL1&2 in pCas9 mit GoTaq® G2 Green Master Mix (Promega) 4.11.9 Konstruktion weiterer Vektoren mit CRISPR-Cas9 Maschinerie aus pCas9_hupL1/2 4.12 Genomeditierung in Rhodobacter sphaeroides 4.12.1 Transformation durch chemische Kompetenz mit PEG-Methode 4.12.2 Transformation durch chemische Kompetenz nach Hanahan et al. (1991) 4.12.3 Konjugation mit Escherichia coli S17-1 4.12.4 Elektroporation 4.12.5 Bioballistische Genomeditierung mit PDS-1000/He Particle Delivers System (BIORAD) 4.12.6 Konjugation mit Escherichia coli S17-1 nach Mougiakos et al. (2019) 65 4.13 Probenvorbereitung für Sequenzierungen 4.13.1 Illumina MiSeq (Genomsequenzierung) 4.13.2 MinION (Genomsequenzierung) 4.13.3 Illumina HiSeq (Transkriptomsequenzierung) 4.14 Bioinformatische Methoden 4.14.1 Genomsequenzierung (Re-Sequenzierung) 4.14.2 Transkriptom-Datenanalyse 5. Ergebnisse und Diskussion 5.1 Schrittweise Reduktion des Vollmediums nach Krujatz et al. (2014) zum Fruchtsaft-Minimalmedium 5.2 Untersuchung der Wasserstoffproduktion in Fruchtsaft-Minimalmedium 5.3 Kontinuierliche Aufzeichnung von Prozessdaten im 1,2 L Bioreaktor 5.3.1 Vergleich der Reaktorläufe in Vollmedium nach Krujatz et al. (2014), Trauben- und Ananas-Minimalmedium der Stämme DSM 158 und SubH2 5.3.2 Prozessgasanalyse 5.4 Analyse des Genoms 5.4.1 Multiples Sequenzalignment der kompletten genomischen Assemblies von Rhodobacter sphaeroides 5.4.2 MiSeq-Sequenzierung des Stammes Rhodobacter sphaeroides 2.4.1. SubH2 5.4.2.1 Bioinformatische Funktionsanalyse von SNPs 5.4.2.2 SNP-Analyse mittels Homology-Modeling 5.4.3 Genomische Architekturanalyse mittels MinION Sequenzierung der Rhodobacter sphaeroides Stämme DSM 158 und 2.4.1. SubH2 5.4.4 Vergleich der MiSeq- und MinION Genomanalysen 5.5 Analyse des Transkriptoms 5.6 Analyse der Genexpression mit qRT-PCR im Vergleich mit der Wasserstoffproduktion 5.7 CRISPR-Cas9 zum Plasmid-basierten hupL Knock-out 5.7.1 Erstellung der Plasmide pCas9_hupL1 und pCas9_hupL2 5.7.2 PEG-basierte Transformation nach Fornari et al. (1982) 5.7.3 Transformation mittels Elektroporation 5.7.4 Erstellung weiterer Vektoren mit CRISPR-Cas9_Maschinerie aus pCas9_hupL1&2 5.7.5 Transformation mittels Konjugation I 5.7.6 Bioballistische Transformation 5.7.7 Problembehandlung zur Transformation 5.7.8 Transformation mittels Konjugation II 6 Zusammenfassung 7 Ausblick 8 Summary Literaturverzeichnis Anhangsverzeichnis Anhang Versicherung