The thesis focuses on the role of Psb28 proteins, namely the Psb28-1 and its homolog Psb28-2, in the biogenesis of the Photosystem II complex (PSII) in the cyanobacterium Synechocystis PCC 6803. The aims of this work were to localize the proteins within the cells, and to determine their function. A fraction of both Psb28 proteins was identified in the monomeric PSII core complexes but most proteins were found in the unassembled protein fraction associated with thylakoid membranes. Psb28-1 was mostly detected as a dimer while Psb28-2 as a monomer. Psb28-1 also differed from Psb28-2 by its higher affinity to the PSII core complex lacking CP43 antenna. Characterization of Psb28-less mutants suggested regulatory function of the proteins in PSII biogenesis in connection with chlorophyll biosynthetic pathway. Analysis of preparations isolated using FLAG-tagged versions of Psb28 proteins showed their association with Photosystem II - Photosystem I supercomplexes, especially under increased irradiance, and supported a role of Photosystem I in the PSII biogenesis.

abstract: The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Made available in DSpace on 2014-07-29T15:01:49Z (GMT). No. of bitstreams: 1 DALESSANDRO_Emmanuel - texto - parte 1.pdf: 1095655 bytes, checksum: 244da2ff7d05e25ed83b393557a7226d (MD5) Previous issue date: 2012-07-12 / The Wastewater Treatment Plant (WWTP) of Trindade (16 ° 39'09'' S and 49 ° 31'50'' W), also known as Barro Preto WWTP, consists of preliminary treatment followed initially by three modules in parallel (A, B and C), each containing one anaerobic pond, one facultative pond and one maturation pond in series (Australian system), being the effluent released in the stream Barro Preto. The WWTP began operating in 1997 and had the final plan in 2011. This study aimed to evaluate treatment efficiency and the module based on algal community structure and physico-chemical variables during a period of six months (September, October, November, December 2010 and April and May 2011), which contains the dry and rainy periods. Were analyzed in reference to the structure and dynamics of algae: biomass, density, richness, diversity, equality, abundance and dominance; for physic-chemical analysis were: flow rate, wastewater temperature, depth, euphotic zone, HDT, pH, DO, CND, TP, PO4 -3, NH4 +, NO3, DOB5 20°C, COD, TSS, FSS, VSS and also climatic variables. The algae were collected through non-selective process (Van Dorn bottle). The samples abiotic, chlorophyll a and pheophytin were processed according to APHA, and the density algal by the Utermöhl method. Principal Component Analysis (PCA) were performed to characterize the physic-chemical processes and the spatial and temporal differentiation. It was also made a Canonical Correspondence Analysis (CCA). The facultative and maturation ponds shown to be influenced by the seasonality of the region, and presented variance in depth. In average, the module A showed an efficiency of COD removal of 45%, and optimal efficiency in removing DBO5 20°C (84%). The facultative pond showed the highest mean of PO4 -3 efficiency removal (48%), NH4 + (36%) NO3 - (44%), BOD5 20°C (74%). OD profile of type clinograde and thermal stratification was recorded in three months of collection. The maturation pond showed higher mean removal efficiency of PT (27%) and COD (19%). In just two months was recorded thermal stratification, and found clinograde profile throughout the study. The ponds presented one of the largest ever recorded phytoplankton densities in stabilization ponds and 170 taxa recorded throughout the study, being 40% of cyanobacteria are potentially toxic. The most abundant species in both ponds and throughout the study were Chlorella vulgaris, Chlorella minutissima, Closteriopsis acicularis, Merismopedia tenuissima, Synechococcus sp and Synechocysitis sp (dominant). The ponds presented according to the indices, low species diversity and low beta diversity. The beta diversity showed that the maturation pond has more flexibility in the rainy season and optional in the dry season. The canonical correspondence analysis (CCA) showed the most abundant species when best developed in seasonality. C. acicularis in the rainy season, C. minutissima in the end of the rainy season and the beginning of the dry season, M. tenuissima in late dry season, C. vulgaris and Synechococcus sp in late dry season and beginning of precipitation. This analysis showed which environmental variables can also influence the in distribution of species. / A Estação de Tratamento de Esgotos (ETE) de Trindade (16°39 09 S e 49°31 50 O), conhecida também como ETE Barro Preto, é composta por tratamento preliminar seguido, inicialmente, por três módulos em paralelo (A, B e C), contendo cada, uma lagoa anaeróbia, uma lagoa facultativa e uma lagoa de maturação em série (sistema australiano), sendo o efluente lançado no córrego Barro Preto. O presente estudo visou avaliar e eficiência de tratamento do módulo A com base na estrutura da comunidade algal e em variáveis físico-químicas, num período de seis meses (setembro, outubro, novembro, dezembro de 2010 e abril e maio de 2011), que contém períodos de estiagem e chuvosos. Foram analisados a estrutura e a dinâmica das algas: biomassa, densidade, riqueza, diversidade, equidade, abundância e a dominância; para as análises físico-químicas foram: vazão, temperatura da água residuária, profundidade, zona eufótica, TDH, pH, OD, CND, PT, PO4 -3, NH4 +, NO3, DBO5 20°C, DQO, SST, SSF, SSV e também variáveis climáticas. As algas foram coletadas através de processo não seletivo (garrafa de van Dorn). As amostras abióticas, clorofila a e feofitina foram processadas de acordo com APHA, e a densidade algal pelo método de Utermöhl. Análises de Componentes Principais (ACP) foram feitas para caracterização dos processos físico-químicos e para a diferenciação espacial e temporal. Também foi feita uma Análise de Correspondência Canônica (ACC), com o intuito de verificar como as espécies mais abundantes se comportaram em relação as variáveis abióticas. A lagoa facultativa e de maturação mostraram influência da sazonalidade da região, além de apresentar variações na profundidade. Em média, o módulo A apresentou eficiência de remoção de DQO de 45%, e eficiência na remoção de DBO5 20°C de 84%. A lagoa facultativa foi a que apresentou maior média de eficiência de remoção de PO4 -3 (48%), NH4 + (36%), NO3 (44%), DBO5 20°C (74%). Registrou-se também perfil de OD do tipo clinogrado e estratificação térmica em três meses de coleta. A lagoa de maturação apresentou maior média de eficiência de remoção de PT (27%) e DQO (19%). Em apenas dois meses foi registrada a estratificação térmica, e verificou-se perfil clinogrado durante todo o estudo. As lagoas apresentaram uma das maiores densidades fitoplanctônicas já registrada em lagoas de estabilização, com 170 táxons durante todo o estudo, e 40% das cianobactérias registradas são potencialmente tóxicas. As espécies mais abundantes em ambas as lagoas e durante todo o estudo foram Chlorella vulgaris, Chlorella minutissima, Closteriopsis acicularis, Merismopedia tenuissima, Synechococcus sp e Synechocystis sp (dominante). As lagoas apresentaram de acordo com os índices, pouca diversidade específica e pouca diversidade beta. A diversidade beta revelou que a lagoa de maturação tem maior flexibilidade no período chuvoso e a facultativa no período de seca. As Análises de correspondência canônica (ACC) mostraram quando as espécies mais abundantes melhor se desenvolveram na sazonalidade. C. acicularis no período chuvoso, C. minutissima na final da chuva e início de seca, M. tenuissima no final da seca, C. vulgaris e Synechococcus sp. em no final da seca e início de chuva. Esta análise mostrou também que variáveis ambientais também podem influenciar na distribuição das espécies.

abstract: Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2018

abstract: Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which was shown by HPLC and protein analysis. Although Δsll1951 was more susceptible to osmotic stress than the wild type, the general viability of the mutant remained unaffected. In a different study a combination of mutants having single or multiple deletions of putative carotenoid cleavage dioxygenase (CCD) genes was created. CCDs are presumed to play a role in the breakdown of carotenoids or apo-carotenoids. The carotenoid profiles of the mutants that were grown under conditions of increased reactive oxygen species were analyzed by HPLC. Pigment lifetimes of all strains were estimated by 13C-labeling. Carotenoid composition and metabolism were similar in all strains leading to the conclusion that the deleted CCDs do not affect carotenoid turnover in Synechocystis. The putative CCDs either do not fulfill this function in cyanobacteria or alternative pathways for carotenoid degradation exist. Finally, slr0941, a gene of unknown function but a conserved genome position in many cyanobacteria downstream of the δ-carotene desaturase, was disrupted. Initially, the mutant strain was impaired in growth but displayed a rather normal carotenoid content and composition, but an apparent second-site mutation occurred infrequently that restored growth rates and caused an accumulation of carotenoid isomers not found in the wild type. Based on the obtained data a role of the slr0941 gene in carotenoid binding/positioning for isomerization and further conversion to mature carotenoids is suggested. / Dissertation/Thesis / Ph.D. Microbiology 2011

abstract: Creating sustainable alternatives to fossil fuel resources is one of the greatest challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

abstract: One solution to mitigating global climate change is using cyanobacteria or single-celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a variety of positive and negative ways that are captured by the model: light intensity (LI) attenuates through a microalgal culture, light absorption provides the energy and electron flows that drive photosynthesis, microalgae pool absorbed light energy, microalgae acclimate to different LI conditions, too-high LI causes damage to the cells’ photosystems, and sharp increases in light cause severe photoinhibition that inhibits growth. The model accounts for all these phenomena by using a set of state variables that represent the pooled light energy, photoacclimation, PSII photo-damage, PSII repair inhibition and PSI photodamage. Sets of experiments were conducted with the cyanobacterium Synechocystis sp. PCC 6803 during step-changes in light intensity and flashing light. The model was able to represent and explain all phenomena observed in the experiments. This included the spike and depression in growth rate following an increasing light step, the temporary depression in growth rate following a decreasing light step, the shape of the steady-state growth-irradiance curve, and the “blending” of light and dark periods under rapid flashes of light. The LI model is a marked improvement over previous light-dependent growth models, and can be used to design and interpret future experiments and practical systems for generating renewable feedstock to replace petroleum. / Dissertation/Thesis / Doctoral Dissertation Civil and Environmental Engineering 2017

Most proteins must fold into native three-dimensional structures to be functional. But, newly synthesized proteins are at high risk of misfolding and aggregating in the cell. Stress, disease or mutations can also cause protein aggregation. A cyanobacterial small heat shock protein, Hsp16.6, can act as a chaperone to prevent irreversible protein aggregation during heat stress. This thesis is focused on the properties of proteins that were associated with Hsp16.6 during heat stress, and which therefore may be “substrates” of Hsp16.6. Bioinformatics were used to determine if Hsp16.6 preferentially binds to proteins with certain properties, and biochemical studies were performed to investigate how the substrates actually behave with Hsp16.6 during heat stress. It was found that Hsp16.6 preferentially binds to proteins with higher molecular weight, higher acidity, higher percentage of charged residues (especially negatively charged residues), and a lower percentage of hydrophobic residues compared to all proteins encoded by the Synechocystis genome. Proteins bound to Hsp16.6 were also slightly enriched in VQL motifs. The potential substrate fructose bisphosphate aldolase class II (FBA) was expressed in E.coli and purified. FBA could be protected by Hsp16.6 from aggregation through forming a complex with Hsp16.6 during heat stress in vitro, consistent with it being a substrate of Hsp16.6. Another potential substrate, elongation factor G1 (EF-G1) was also expressed in E.coli and purified. EF-G1 did not form insoluble aggregates even at 47°C, but circular dichroism spectroscopy revealed the secondary structure has melted at this temperature, and the protein eluted earlier than unheated protein on size exclusion chromatography. Thus, EF-G1 appears heat sensitive, and may also be an in vivo substrate of Hsp16.6. Lastly, in vivo study studies were performed to determine the amount of FBA and EF-G1 in Synechocystis cells. Both proteins are abundant, with FBA levels (around 2% of total cell protein) being about twice that of EF-G1. Further in vivo experiments will be needed to confirm that FBA and EF-G1 are substrates of Hsp16.6.

In der vorliegenden Arbeit wurde die Funktion der beiden cyanobakteriellen Phytochrome Cph1 und Cph2 untersucht. Dafür wurde zunächst das Wachstum von Mutanten mit einem inaktivierten cph1- bzw. cph2-Gen unter verschiedenen Lichtbedingungen analysiert. Das Wachstum aller Phytochrommutanten war unter Starklicht beeinträchtigt. Dahingegen wuchs die cph1-Mutante im FRL schlechter als der Wildtyp, während das Wachstum der cph2-Mutante im RL vermindert war. Eine cph1/cph2-Doppelmutante zeigte unter allen Lichtbedingungen eine Wachstumsreduktion, die der jeweiligen Einzelmutante ähnlich war. Die genaue Ursache für die Beeinträchtigung des Wachstums der Phytochrommutanten konnte nicht ermittelt werden. Die verschiedenen Aspekte der Photosynthese, wie Pigmentzusammensetzung, maximale Netto-Sauerstofffreisetzungsrate und 77K-Fluoreszenzemission, waren in den Phytochrommutanten nicht signifikant verändert. Bei Synechocystis sp. PCC 6803 konnte eine lichtgerichtete Bewegung beobachtet werden, wobei man aufgrund von Aktionsspektren der Motilität eine Funktion der Phytochrome oder phytochromähnlichen Proteine bei der Steuerung der phototaktischen Bewegung vermutet. Dem Cph1-Protein konnte hierfür keine Rolle zugeordnet werden. Dahingegen scheint der Photorezeptor Cph2 die lichtgerichtete Bewegung der Zellen in Richtung einer Blaulichtquelle zu inhibieren. Eine Interaktion mit einem klassischen Blaulicht-Rezeptor konnte ausgeschlossen werden. / The function of the cyanobacterial phytochromes Cph1 and Cph2 was investigated. At first, the growth of mutants with an inactivated cph1 or cph2 gene was analysed under different light conditions. The growth of all phytochrome mutants was affected under high-light conditions. However, the cph1 mutant grew slower than the wild type in far-red light, whilst the cph2 mutant revealed a reduced growth under red light conditions. A decreased growth of the cph1/cph2 double mutant was observed under all light conditions with a growth rate similar to the corresponding single mutant. The exact reason for the growth impairment of the phytochrome mutants could not be ascertained. Different aspects of photosynthesis (pigment composition, maximal net-oxygen evolution and 77K fluorescence emission) were not changed significantly in the phytochrome mutants. Synechocystis sp. PCC 6803 shows a movement towards a light source. Based on action spectra of motility phytochromes and phytochrome-like proteins are supposed to have a function in regulating the phototactic movement. An influence of the Cph1 protein in the phototactic movement was not demonstrated. Whereas, the Cph2 protein seems to be involved in the inhibition of the cell movement towards blue light. An interaction with a typical blue-light receptor was excluded.

Der Abbau der PBS unter Stickstoffmangel ist in Cyanobakterien ein ubiquitärer Mechanismus. Essenziell für den Abbau ist das Protein NblA. Das in Nostoc sp. PCC 7120 gut untersuchte ~7 kDa große Protein interagiert als Homodimer mit den PBS und dem Chaperon ClpC. Soweit bekannt kodieren alle Cyanobakterien ein essenzielles NblA-Protein. Der Modellorganismus Synechocystis sp. PCC 6803, dagegen kodiert mit NblA1 und NblA2 sogar zwei für den PBS-Abbau entscheidende Proteine. In dieser Arbeit konnte erstmalig durch in vitro und in vivo Interaktionsstudien mithilfe von pull down-Versuchen und FRET gezeigt werden, dass NblA1 und NblA2 als biologisch aktive Form ein Heterodimer bilden. In vitro-pulldown-Versuche zeigten für Synechocystis eine Interaktion des Heterodimers mit den PBS und ClpC. Durch die Ausbildung dieses ternären Komplexes markiert NblA1/NblA2 die PBS für den Abbau durch eine Clp-Protease mit ClpC als Chaperonpartner. In Photobionten gibt es eine Reihe von clp-Genen, so besitzen Cyanobakterien vier proteolytische clp Untereinheiten. Aus diesen bilden sich gemischte Heptamere aus je zwei Clp-Untereinheiten. Zwei lösliche, im Cytoplasma vorkommende Proteasen wurden bis jetzt zweifelsfrei identifiziert, eine dritte, an der Thylakoidmembran assoziierte Protease wird außerdem vermutet. Diese putative Protease, vermutlich aus dem Chaperon ClpC und den proteolytischen Untereinheiten ClpP1 und ClpR aufgebaut, wurde heterolog exprimiert. Mittels Koreinigung konnte ein funktioneller proteolytischer Kern gereinigt werden, der zusammen mit dem Chaperon ClpC eine aktive Clp-Protease bildet. Durch Größenausschlusschromatografie und Untersuchungen zur Proteaseaktivität konnte diese dann erstmalig charakterisiert werden. Des Weiteren zeigten in vitro-Degradationsversuche mit der aktiven Protease zweifelsfrei, dass das NblA1/NblA2-Heterodimer durch ClpC-ClpP1/ClpR abgebaut wird. Der Abbau von NblA1/NblA2 kann demnach auch ohne ein Substrat durch die Protease erfolgen. / The degradation of pycobilisomes under nitrogen deficiency, which is visible as a color change from blue green to yellow green, is an ubiquitary mechanism. Essential for the degradation is the small NblA protein. The well characterized NblA protein from Nostoc sp. PCC 7120 builds as biological active form a homodimer and interacts with the phycobilisomes and ClpC. However, the model organism Synechocystis sp. PCC 6803 possess two nblA genes, nblA1 and nblA2, which are both essential for phycobilisome degradation. In this work it could be shown by interaction studies and Förster resonance energy transfer that NblA1 and NblA2 builds as biological active form a heterodimer. Furthermore it could be shown that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease. Clp proteases of Cyanobacteria consist, besides the chaperones ClpC and ClpX of three different proteolytic subunits (ClpP1, 2, 3) and the ClpP variant ClpR which lacks the catalytic triad typical of Serine-type proteases. In Synechococcus 7942 two Clp proteases could be identified by gel filtration and immunoblot analyses, which provided evidence that each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one core consisting of ClpP1 and ClpP2, and the other of ClpP3 and ClpR, in which subunits ClpP1/ClpP2 interact with ClpX and the ClpP3/ClpR protease with ClpC. In addition to these two soluble proteases, a third, membrane associated proteolytic complex is assumed, consisting of ClpP1 and ClpR. In Synechocystis 6803, homologs of the Clp machinery could be found by BLAST analyzes. This putative ClpP1/ClpR protease could be characterized in vitro by size-exclusion chromatography and degradation assays. Furthermore it could be shown a degradation of the NblA1/NblA2 heterodimer by the protease.