Modulare Polyketidsynthasen (PKS) sind Multienzymsysteme des bakteriellen Sekundärstoffwechsels. An ihnen läuft eine schrittweise Biosynthese vielfältiger Kohlenstoff-Gerüste ab, die von einfachen Carbonsäure-Einheiten ausgeht. Polyketid-Verbindungen zeigen eine große Bandbreite pharmazeutisch interessanter Aktivitäten. In dieser Arbeit wurde eine Reihe von Evolutionsstudien durchgeführt. Zunächst wurden die phylogenetischen Beziehungen zwischen modularen PKS und anderen PKS-Systemen sowie Fettsäuresynthasen untersucht, wodurch ihre zentrale Stellung innerhalb eines langen Evolutionsprozesses gezeigt werden konnte. Eine detaillierte Analyse der Phylogenien von Domänen bakterieller modularer PKS ergab, dass das Ausmaß an Genduplikationen, Genverlusten und Ereignissen horizontalen Gentransfers zwischen den verschiedenen Bakteriengruppen beträchtlich variiert. Aus der Genomsequenz des Actinobakteriums Streptomyces avermitilis wurden die Phylogenien aller Domänentypen rekonstruiert. Der Vergleich dieser Einzelphylogenien ermöglichte es, eine Reihe von homologen Rekombinationsereignissen aufzufinden. Homologe Rekombination scheint der Hauptmechanismus zu sein, auf dem die Strukturvielfalt der Polyketide in Bakterien beruht. Mit Hilfe eines „genome mining“-Ansatzes konnte im Genom des Cyanobakteriums Nostoc punctiforme eine Reihe von Biosynthese-Clustern, die zu den PKS und nichtribosomalen Peptidsynthetasen gehören, identifiziert werden. Durch chromatographische und massenspektrometrische Analysen von Zellextrakten und Kulturüberständen konnten einige der Biosynthese-Cluster bestimmten Metaboliten zugeordnet werden. Eines der Cluster wurde hinsichtlich des produzierten Metaboliten und der Regulationsstruktur eingehender charakterisiert. Die Folgerungen aus den gewonnen Ergebnissen werden im allgemeinen Zusammenhang der Evolution metabolischer Diversität ausführlich diskutiert. / Modular polyketide synthases (PKS) are multienzym systems of bacterial secondary metabolism. They perform a stepwise biosynthesis of diverse carbon skeletons from simple carboxylic acid units. Polyketide compounds possess a wide range of pharmaceutically interesting activities. In this study, a series of evolutionary analyses was performed. Initially, the phylogenetic relationships between modular PKS and other PKS systems as well as fatty acid synthases were investigated revealing their central position within a long evolutionary process. In detail reconstruction of the phylogenies of bacterial modular PKS domains demonstrated that the extent of gene duplications, gene losses and horizontal gene transfer events varies considerably between different bacterial groups. Using the genome sequence of the actinobacterium Streptomyces avermitilis the phylogenies of all domain types were reconstructed. Comparison of these phylogenies allowed for detecting numerous events of homologous recombination, which appears to be the main mechanism underlying polyketide structural diversity in bacteria. A genome mining approach revealed a number of biosynthesis clusters of the PKS and nonribosomal peptide synthetase type in the genome of the cyanobacterium Nostoc punctiforme. Cell extracts and culture supernatants were analysed by means of liquid chromatography and mass spectrometry and some of the biosynthesis clusters could be assigned to specific metabolites. One of the clusters was characterised in greater detail regarding the produced metabolite and the cluster’s regulatory structure. The implications of the results are extensively discussed within the general context of the evolution of metabolic diversity.

<p>U radu je ispitana zastupljenost azotofiksirajućih cijanobakterija, ukupnog broja algi i ukupnog broja bakterija u različitim tipovima zemlji&scaron;ta na jedanaest lokaliteta u Vojvodini, od čega se sedam nalaze u za&scaron;tićenim prirodnim dobrima. Ispitana je brojnost u zavisnosti od dubine pedolo&scaron;kog profila, kao i od godi&scaron;njeg doba. Izvr&scaron;ena je izolacija sojeva azotofiksirajućih cijanobakterija, određena njihova taksonomska pripadnost i osnovne citolo&scaron;ke karakteristike. Ispitana je ultrastruktura vegetativnih ćelija, heterocista i spoljnih struktura na ćelijama fimbrije i pili, transmisionim elektronskim mikroskopom. Izvr&scaron;ena je genetička karakterizacija izolovanih sojeva azotofiksirajućih cijanobakterija PCR metodom analizom STRR fragmenata DNA.<br />Brojnost azotofiksirajućih cijanobakterija i ukupna brojnost algi je bila znatno veća kod hidromorfnih i halomorfnih zemlji&scaron;ta, nego kod automorfnih zemlji&scaron;ta. Najveća prosečna godi&scaron;nja brojnost azotofiksirajućih cijanobakterija, u povr&scaron;inskom sloju 0-5cm, je utvrđena kod zemlji&scaron;ta fluvisol u SRP &ldquo;Koviljsko petrovaradinskom ritu&rdquo;, 150864 jedinki po gramu apsolutno suvog zemlji&scaron;ta. Zemlji&scaron;te sa najnižom brojno&scaron;ću azotofiksirajućih cijanobakterija, je gajnjača u NP Fru&scaron;ka gora, 1582 jed./gr zemlji&scaron;ta u povr&scaron;inskom sloju.<br />Kod svih ispitivanih zemlji&scaron;ta brojnost azotofiksirajućih cijanobakterija je bila najveća u povr&scaron;inskom sloju zemlji&scaron;ta, 0-5 cm dubine, opadala je sa dubinom zemlji&scaron;ta i bila najmanja u najdubljem sloju, 30 - 60 cm. Kod najvećeg broja ispitivanih zemlji&scaron;ta brojnost azotofiksirajućih cijanobakterija je bila najveća tokom zimskog perioda. Azotofiksirajuće cijanobakterije su dominantne u na&scaron;im zemlji&scaron;tima i zastupljene sa 56.27% u odnosu na druge grupe algi.Izolovano je 30 sojeva azotofiksirajućih cijanobakterija: 19 sojeva Nostoc-a, 4 soja Anabaena, 4 Cylindrospermum, i po jedan soj Calothrix, Tolypothrix i Phormidium. Prosečna zastupljenost heterocista, ćelija koje vr&scaron;e azotofiksaciju, kod roda Nostoc je iznosila 8.28%, Anabaena 4.25%, Cylindrospermum-a 2.93%, Calotrix elenkinii 6.19% i Tolypothrix 7.76%.</p><p>Ultrastrukturnim ispitivanjem, TEM mikroskopom, vegetativnih ćelija azotofiksirajućih cijanobakterija uočili smo inkluzije redovnog pojavljivanja: karboksizome (Cs), cijanoficinkse granule (CG), polifosfatne granule (PG), ribozome (R), lipidne granule (&szlig; &ndash;granule) i tilakoide (T), kao i inkluzije neredovnog pojavljivanja: membranom ograničene kristalne inkluzije.<br />Koristeći TEM i tehniku bojenja ćelija sa RR i ultratankih preseka utvrdili smo prisustvo omotača od fimbrija kod tri soja (A.314, A.azollae i N.302) i tipične fimbrije kod dva soja (N.311 i N.9229). Metodom negativnog bojenja NS PTA uočili smo takođe tipične fimbrije, igličastog-dlakastog izgleda, jasnih granica niti kod tri soja (N.302, N.7901 i N.9229), međutim uočili smo i atipične sluzne fimbrije, koje nemaju jasno izražene granice, ali su veoma moćno ra&scaron;irene oko vegetativnih ćelija, kod tri soja (A.314, A.azollae, N.311).<br />Kod simbiotskih-infektivnih sojeva N.7901 i N.9229 javljaju se samo tipične fimbrije iz prve klase, a kod diazotofnih sojeva i simbiotskog - neinfektivnog soja A.azollae javljaju se atipične-sluzne fimbrije iz druge klase.<br />Za ispitivanje sličnosti cijanobakterija metodom PCR-a pomoću STRR konzervativnih sekvenci DNA genoma kori&scaron;ćeno je 39 sojeva azotofiksirajućih cijanobakterija i kod 38 je utvrđeno njihovo prisustvo. Svi sojevi se mogu podeliti u tri grupe, klastera. Prvi klaster je najveći i obuhvata 24 soja i deli se na dva podklastera: Ia koji obuhvata 12 sojeva gde dominiraju sojevi Nostoc-a (8), i podklaster Ib koji obuhvata takođe 12 sojeva, od čega 6 sojeva pripada rodu Anabaena. Podklaster Ia i podklaster Ib pokazuju različitost od 90%. Sva tri simbiozna, infektivna, soja Nostoc-a se nalaze u klasteru I: N.7901, N.9229 i N. 8001. Svaki simbiozni soj Nostoc-a ima genetske sličnosti sa po jednim diazotrofnim sojem Nostoc-a izolovanim iz zemlji&scaron;ta Vojvodine.<br />Klaster II obuhvata sedam (7) sojeva među kojima dominiraju sojevi Cylindrospermum-a, dok klaster III obuhvata 7 sojeva od čega 6 pripadaju rodu Nostoc, a jedan rodu Rivularia.Detaljno poznavanje svojstava izolovanih azotofiksirajućih cijanobakterija doprineće njihovoj budućoj primeni kako u proizvodnji ratarskih i povrtarskih kultura, tako i u biotehnolo&scaron;koj proizvodnji</p> / <p>In this study examined is the frequency of nitrogen-fixing cyanobacteria, total number of algae and total number of bacteria in different soil types on eleven localities in the Vojvodina Province. Seven out of those eleven localities are found in protected nature reserves. Actually, studied was the number of the cyanobacteria and algae depending on the depth of pedological characterization as well as on season. First, isolated were the types of nitrogen-fixing cyanobacteria, determined was their taxonomic origin and basic cytological characteristics. Also examined was the ultrastructure of vegetative cells, heterocysts and other outer structures on fimbriae and pili cells using TEM, transmission electron microscope. Finally, performed was the genetic characterization of isolated types of nitrogen-fixing cyanobacteria using the PCR method and analyzing STRR fragments of DNA.<br />The presence of nitrogen-fixing cyanobacteria and total number of algae was significantly higher with hydromorphic and halomorphic soils than with authomorphic ones. Highest annual average number of nitrogen-fixing cyanobacteria in the topsoil (0-5 cm) was reported with fluvisol soil in Special Nature Reserve &bdquo;Koviljsko petrovaradinski rit&rdquo; (Swamp) and there were 150864 units of bacteria per gram of absolutely dry soil. The soil with the lowest presence of nitrogen-fixing cyanobacteria recorded was cambisol in National Park &ldquo;Fruska gora&rdquo; with 1582 units per gram of soil in the topsoil.<br />With all the researched types of soils the number of nitrogen-fixing cyanobacteria was in the topsoil, 0-5 cm of depth and decreased in line with the depth<br />of soil and lowest was at the deepest layer, 30-60cm. The highest frequency of nitrogen-fixing cyanobacteria was found during the winter season with most of the examined soils. Nitrogen-fixing cyanobacteria are the dominant type of bacteria in our soils and are presented with 56, 27% compared to other types of algae.<br />30 strains of nitrogen-fixing cyanobacteria were isolated: 19 types of Nostoc sp., 4 of Anabaena sp. and one in each genus of Calothrix, Tolypothrix and Phormidium.<br />Using ultrastructural examination and TEM microscope when studying vegetative cells of nitrogen-fixing cyanobacteria observed were the inclusions of regular frequency: carboxysomes (Cs), cyanophycin granules (CG) , polyphosphate granules (PG), ribosomes (R), lipid granules (SS -granule ) and thylakoids ( T ) as well as the inclusion of irregular occurrence: a membrane-bound crystal inclusions.<br />Using TEM technique and staining the cells with the RR and ultra thin cross section, we determined the presence of depletion of the fimbriae with three strains (A.314, A.azollae and N.302) and typical fimbriae with the two strains (N.311 and N.9229). Applying the method of negative staining NS PTA also noticed were a typical fimbriae, needle-hairy like looks with clear boundaries with the three strains (N.302, N.7901, N.9229). However, also observed were atypical mucous fimbriae, which do not have clearly expressed borders, but they are very strongly spread around the vegetative cells, with the three strains (A.314, A.azollae, N.311).<br />With symbiotic - infective strains N.7901 and N.9229 only typical fimbriae of first class occurred, and in diazotroph strains and symbiotic &ndash; non infectious strain A. azollae found were atypical mucous fimbriae of second class.<br />To test the similarity of cyanobacteria by PCR method and using a STRR - conservative DNA sequence of the genome used were 39 strains fixing cyanobacteria and with 38 established was their presence. All strains can be divided into three groups of clusters. The first cluster is the largest and covers 24 strains, and is divided into two subclusters: Ia which includes 12 strains,where predominant are Nostoc strains ( 8 ) , and subcluster Ib , which also implies 12 strains , out of which 6 strains belong to the genus Anabaena. Subcluster Ia and Ib show a difference of 90 %. All three symbiotic , infectious Nostoc strains are classified in a cluster I: N.7901, N.9229 and N. 8001. Each symbiotic Nostoc strain has a genetic similarity with one di-nitrogen Nostoc strain isolated from a lot of different soils in Vojvodina.<br />Cluster II includes seven (7) strains , including strains among which the predominant are Cylindrospermum ones , while cluster III includes 7 strains of which 6 belong to the genus Nostoc and one to genus Rivularia.<br />Detailed knowledge of the properties of isolated fixing cyanobacteria could contribute to their future application both in the production of field crops and vegetables, as well as in biotechnological production.</p>

Some cyanobacteria strains have biofertilization and bioconditioning effects in soils. The objective of this study was to identify cyanobacteria with potential to improve the N fertility and structural stability of degraded soils and evaluate their effectiveness in soils of the Eastern Cape, South Africa. Isolation and characterization of the indigenous cyanobacteria strains with desirable properties was first to be undertaken because their effects are known to differ from strain to strain. Cyanobacteria strains 3g, 3v, and 7e were identified from 97 strains isolated from selected soils. Nostoc strains 3g and 3v had greater ability to produce exocellular polysaccharides (EPS) but low potential to fix atmospheric N2 (4.7 and 1.3 nmol C2H4 μg chl-1 h-1, respectively). On the other hand, strain 7e had the highest capability to fix atmospheric N2 (16.1 nmol C2H4 μg chl-1 h-1) but had the least ability to produce EPS. Evaluation of the strains was done in glasshouse studies starting with Nostoc strain 9v isolated from a Tanzanian soil, followed by the indigenous strains isolated from soils in Hertzog and Qunu, South Africa. Inoculation was done by uniformly applying cyanobacteria on the surface of potted soils at a rate of 6 g m-2. First harvest and soil sampling took place after six weeks, and the top 25 mm of the soil was mixed, replanted, and sampled again after a further six weeks (second harvest). Inoculation with Nostoc strain 9v increased soil N by 40 percent and 17 percent in Guquka and Hertzog soils, respectively, and consequently increased maize dry matter yields by 40 and 49 percent. Soil C increased by 27 percent and 8 percent in Guquka and Hertzog soils, respectively, and this increase was significantly associated with that of soil N (R2 = 0.838). Higher contents of soil C, soil N and mineral N, however, were found in non-cropped soils. Scanning Electron Microscopy (SEM) revealed coatings of EPS on soil particles and fragments of non-cropped inoculated soils, with iii other particles enmeshed in networks of filaments, in contrast to cropped and/or non-inoculated soils. The proportion of very stable aggregates was increased by inoculation but cropping with maize reduced the aggregate stability. Inoculating Hertzog soil with indigenous strains 3g and 7e increased the nitrate N in the first cropping by 49 percent and 69 percent respectively, in cropped soils. In the second cropping increases in mineral N were 41 percent and 43 percent in 3g and 7e inoculated soils, respectively. Maize dry matter yields were higher on inoculated soils both in the first and second harvest in response to the improved N status of the soil. Increases in aggregate MWD in cropped soil as determined by fast wetting, mechanical breakdown and slow wetting were 85 percent, 33 percent, 33 percent, respectively, for 3g inoculation, 64 percent, 41 percent, and 41 percent, respectively, for 7e inoculation and 60 percent, 24 percent, 50 percent for inoculation with 9v. In non-cropped soil, increases in MWD as determined by fast wetting, mechanical breakdown and slow wetting were 11 percent, 0 percent, 7 percent, respectively for 3g inoculation, 21 percent, 11 percent, and 7 percent, respectively for 7e inoculation, and 25 percent, 36 percent, and 19 percent for strain 9v inoculation. Scanning electron microscopy observations, which were confirmed by chemical results, revealed that inoculated soils had high EPS and filaments that encouraged soil aggregation and improved aggregate stability. Results of this study show that cyanobacteria strains isolated and selected for their ability to fix atmospheric N2 and produce EPS improved the fertility status and aggregate stability of degraded soils from South Africa.

While much is known about the structure and interactions of the β-carboxysomal proteins, interactions of the proposed vertex protein CcmL with the other components have not yet been directly demonstrated. A fluorescence resonance energy transfer based method combined with other complementary spectrophotometry techniques as well as x-ray crystallography and transmission electron microscopy was used in a Thermosynechococcus elongatus BP-1 model to study these interactions. CcmL was found to interact with the various CcmK shell proteins with a clear binding preference for CcmK2; the previously proposed interaction of CcmL with CcmM was shown to not occur in vitro, and a possible CcmL-CcmL interaction was observed tentatively. In addition, analysis of a novel x-ray crystal structure of Nostoc sp. PCC7120 CcmL in a decameric form suggests a possibility of a CcmL-CcmL back-to-back interaction. This study gives the first direct experimental evidence for the biological role of CcmL as the carboxysomal vertex protein.

No description available.

No description available.

No description available.

No description available.

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>

Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated. Nostoc punctiforme PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by hupSL. The transcription of hupSL was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in N. punctiforme, the hypFCDEAB operon. These genes were found to be situated upstream of hupSL in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon. The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of N. punctiforme was constructed, where hupL was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the hupL- mutant and in wild type N. punctiforme was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup- mutant of the related strain Nostoc PCC 7120 were also studied. This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.