Synechococcus elongatus PCC 7942 is a model organism used to study the circadian rhythm, a process that is driven by an endogenous biological clock that can be modulated by external cues such as light and temperature. Some proteins have been identified that are involved in circadian signal transduction in S. elongatus. Of them, KaiA, KaiB and KaiC comprise the central oscillator components, which are essential for internal timekeeping. SasA is an important protein in the output pathway, which passes the information from central oscillator to downstream components, and thus controls metabolic and behavioral processes. CikA is a major component in the input pathway, which maintains synchrony of the oscillator with the environment. CikA is an unusual phytochrome-like histidine protein kinase. It has a pseudo receiver domain which can not accept a phosphoryl group. CikA is thought to be located at the poles of the cell through interaction between PsR and some protein or protein complex that is also localized at the poles. One of the potential CikA-interacting proteins identified through a yeast two hybrid screen is called hypothetical protein 1. It specifically recognizes a PsR bait in a yeast two hybrid assay. A bioinformatics analysis showed that there are predicted signal peptide and transmembrane domains at the N-terminal and a cytochrome C homolog domain at the C-terminal of Hyp1. Elucidating the features and function of Hyp1 will provide us with useful information to understand the function and working mechanism of CikA, and therefore will help us to clarify the signal transduction in the clock. In this research, I used genetic, cell biological and biochemical approaches to study the features and function of this newly identified clock component Hyp1. To confirm the interaction between PsR and Hyp1 and complement the yeast two hybrid data, I truncated Hyp1 (Thyp1) and purified soluble Thyp1. At the same time, I obtained purified PsR. I tried to copurify the PsR and 6-histidine-tagged Hyp1 on a nickel affinity column. However, PsR non-specifically bound to the column, which eliminated the utility of this approach to study their interaction. In addition to using a biochemical approach to study Hyp1, I constructed three hyp1 overexpression alleles for genetic analysis and two hyp1-yfp overexpression fusion alleles for subcellular localization studies. All of them will help us to understand the features and function of Hyp1.

Bochum, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Bochum, Universiẗat, Diss., 2003.

Techn. University, Diss., 2004--Berlin.

Techn. University Diss., 2005--Berlin.

Cyanobacteria are photosynthetic prokaryotes that live in aquatic environments. The cyanobacterium Synechococcus elongatus PCC 7942, (hereafter S. elongatus) coordinates its day and night behaviors via a circadian clock. The clock is entrained by light/dark cycles but continues to run in constant light conditions. The core circadian clock in S. elongatus is encoded by post-translational modifications of three Kai proteins, but the extent and mechanism of circadian gene expression are unknown. We provide the first unbiased characterization of circadian gene expression in S. elongatus, demonstrating that \(\sim 65\%\) of genes display oscillation in continuous light conditions, with some genes peaking in expression at subjective dawn and others at subjective dusk. We next sought to identify the mechanism by which such a large fraction of the genome could be rhythmically controlled. Through bioinformatic, correlative, and perturbation experiments, we find that circadian changes in chromosome topology/supercoiling are sufficient to drive rhythmic expression (Chapter 2). To further investigate how chromosome topology can control gene expression we performed a high resolution characterization of transcripts and RNA polymerase across the S. elongatus genome (Chapter 3). Bioinformatic analysis of transcription start sites suggests that the AT/GC content a particular region of the promoter is informative in defining the phase at which a transcript is maximally expressed. We find that these sequences are sufficient to drive circadian gene expression at a particular phase and that mutation of single nucleotides in this region can reverse the expression phase of a transcript (Chapter 4). To understand the role of chromosome dynamics in circadian gene expression and cyanobacterial physiology, we tagged and followed chromosomes over multiple cell divisions. We find that S. elongatus cells harbor multiple ordered copies of a single chromosome, and the organization of chromosomes in the cytoplasm facilitates equal segregation of chromosomes to daughter cells (Chapter 5).

Las cianobacterias, microorganismos que realizan la fotosíntesis oxigénica, tienen que adaptar su metabolismo a los distintos retos ambientales a los que se enfrentan, como la limitación de nutrientes o los ciclos de luz oscuridad. Para ello, han desarrolado una serie de mecanismos de gran complejidad y alta regulación que les permiten adaptarse y sobrevivir. En este contexto, PipX, una pequeña proteína exclusiva de cianobacterias, descubierta por el grupo de investigación de genética cianobacteriana de la Universidad de Alicante, actúa como conexión, dependiente del estatus carbono/nitrógeno, entre la proteína de transducción de señales, PII, y el regulador transcripcional, NtcA. Recientemente se han descubierto otras parejas de interacción de PipX, entre las que destaca PipY, miembro de la familia de proteínas de unión a piridoxal fosfato (PLPBP) que forma un operón con PipX en la mayoría de cianobacterias, y la GTPasa de ensamblaje de ribosomas, EngA. La mayoría de estos descubrimientos se han realizado en el organismo modelo, Synechococcus elongatus PCC7942, el cual ha sido el principal objeto de estudio en esta Tesis, centrada fundamentalmente en PipX y sus parejas de interacción. Entre las aportaciones novedosas de estas Tesis se encuentran: 1. La propuesta del empleo de PipY como modelo para el estudio de miembros de la familia PLPBP/COG325; 2. La caracterización de los fenotipos de sobreexpresión de PipX y PipY, dando lugar al descubrimiento de nuevas funciones (formación de polifosfatos) y conexiones entre ellas; 3. La demostración de la existencia de interacción funcional entre EngA y PipX, y la descripción de la función de EngA en el estrés redox en cianobacterias; 4. El avance en el estudio de los terminantes moleculares de la toxicidad de PipX en ausencia de PII y el papel de esta última en el mantenimiento de los niveles intracelulares de PipX. En conclusión, esta Tesis amplía el conocimiento sobre la compleja regulación de los sistemas cianobacterianos en respuesta a distintos estímulos ambientales y, en concreto, las conexiones y el papel de PipX junto a sus antiguas y nuevas parejas de interacción.

A sustainable and low-cost system, namely a photo-bioelectrochemical system (photo-BES), based on the natural blueprint of photosynthetic microorganisms was studied. The aim of this research work is to improve the efficiency of electron transfer of the microorganisms for bioelectricity generation. The first strategy adopted was the evaluation of the exoelectrogenic activity of oxygenic photosynthetic cyanobaterium, Synechococcus elongatus PCC 7942, in biophotovoltaic (BPV) platforms through a comparative performance analysis of bioanode materials. The second approach involved improving the performance of anoxygenic photosynthetic bacterium, Rhodopseudomonas palustris ATCC® 17001™, by varying the ratio of nitrogen to carbon sources (N:C) to maximise both biohydrogen production and exoelectrogenesis for conversion into bioelectricity in photosynthetic microbial fuel cells (photoMFCs). A linear correlation was obtained between average surface roughness/surface area and maximum power density of ITO-coated and graphene/ITO-coated substrates. Graphene/ITO-coated PET bioanodes produced the highest maximum power output of 29±4 μW m-2 in a single chamber BPV device due to improved biofilm formation and improved electrochemical activity. XG Leaf®, also known as graphene paper, helped to bridge the shortcomings of carbon fibres in terms of wettability. The most hydrophilic, 240 μm thick graphene paper, produced the highest maximum power output of 393±20 μW m-2 in a membrane electrode assembly (MEA)-type BPV device, mainly due to reduced electrochemical polarisation. A proof of concept study compared the performance of screen-printed graphene onto a membrane separator against 3D-printed bioanodes coated with carbon nanotubes. One mm thick 3D-printed bioanode was better performing as its structures promoted a much denser biofilm with extensive fibrous extracellular matrix. Using a ratio of N:C=0.20 resulted in higher biohydrogen production and higher exoelectrogenic activity, generating a maximum power output of 361±157 mW m-2 and 2.39±0.13 mW m-2, respectively. This study provided additional insight in improving the electron transfer efficiency, which could be used to further optimise photo-BESs as part of future research and development for sustainable technologies.

PipX es una proteína única de cianobacterias identificada por su habilidad para interactuar de forma excluyente con PII y NtcA, dos reguladores clave implicados en la integración de señales de nitrógeno/carbono y energía, estableciendo un vínculo mecanístico entre ambos. Sin embargo, los datos recabados indican que el papel de PipX no se limita únicamente a esto, sino que parece formar parte de una red de señalización más amplia exclusiva de cianobacterias. La formación de los complejos PII-PipX no solo permite el secuestro de PipX, impidiendo la co-activación de NtcA, sino que favorece la formación de los complejos con el regulador transcripcional cianobacteriano PlmA. Esta interacción con PII afecta a la localización dinámica de PipX en respuesta al estado energético de la célula, limitando su actividad a condiciones de alta energía. Además, PipX está funcionalmente conectado con pipY, el gen aguas abajo que codifica a un miembro de la familia de proteínas PLPBP, universalmente distribuidas e implicadas en la homeostasis de la vitamina B6 y amino ácidos y, en humanos, epilepsia dependiente de vitamina B6. No solo PipX sería capaz de controlar a nivel traduccional la expresión de PipY, sino que ambos participarían en el control de la expresión génica y vías metabólicas comunes. Por último, el análisis de genomas cianobacterianos posiciona a PipX en una red de interacción con PipY y otras 4 proteínas con las que PipX podría tener una relación funcional.

The current investigation is aimed at the reconstruction and analysis of genome-scale metabolic models. Specifically, it is focused on the use of mathematical-computational simulations to predict the cellular metabolism behavior towards bio-products production. The photosynthetic cyanobacterium Synechococcus elongatus PCC7942 was studied as biological system. This prokaryotic has been used in several studies as a biological platform for the synthesis of several substances for industrial interest. These studies are based on the advantage of autotrophic systems, which basically requires light and CO2 for growth. The main objective of this thesis is the integration of different types of biological information, whose interaction can be extract applicable knowledge for economic interests. To this end, our study was addressed to the use of methods for modeling, analyzing and predicting the behavior of metabolic phenotypes of cyanobacterium. The work has been divided into chapters organized sequentially, where the starting point was the in silico metabolic reconstruction network. This process intent to join in a metabolic model of all chemical reactions codified in genome. The stoichiometric coefficients of each reactions, can be arranged into a sparse matrix (stoichiometric matrix), where the columns corresponds to reactions and rows to metabolites. As a result of this process the first model was obtained (iSyf646) than later was updated to another (iSyf714). Both were generated from data ¿omics published in databases, scientific reviews as well as textbooks. To validate them, each one of the stoichiometric matrix together with relevant constraints were used by simulation techniques based on linear programming. These reconstructions have to be flexible enough to allow autotrophic growth under which the organism grows in nature. Once the reconstructions were validated, environmental variations can be simulated and we were able to study its effects through changes in outline system parameters. Subsequently, synthetic capabilities were evaluated from the in silico models in order to design metabolic engineering strategies. To do this a genetic variation was simulated in reactions network, where the disturbed stoichiometric matrix was the object of the quadratic optimization methods. As a results sets of optimal solutions were generated to enhanced production of various metabolites of energetic interest such as: ethanol, n-butanol isomers, lipids and hydrogen, as well as lactic acid as the compound which is an interest to the industry. Furthermore, functionally coupled reactions have been studied and have been weighted to the importance in the production of metabolites. Finally, genome-scale metabolic models allow us to establish criteria to integrate different types of data to help of find important points of regulation that may be subject to genetic modification. These regulatory centers have been investigated under drastic changes of illumination and have been inferred operational principles of cyanobacterium metabolism. In general, this thesis presents the metabolic capabilities of photosynthetic cyanobacterium Synechococcus elongatus PCC7942 to produce substances of interest, being a potential biological platform for clean and sustainable production. / Triana Dopico, J. (2014). Model-based analysis and metabolic design of a cyanobacterium for bio-products synthesis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/39351 / TESIS