POPULARITY
Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 03/05
Diese Arbeit behandelt verschiedene ultraschnelle photochemische Prozesse. Ein Schwerpunkt liegt dabei auf der Charakterisierung der perizyklischen Reaktionen photochromer Indolylfulgide und -fulgimide sowie der Untersuchung verschiedener Möglichkeiten der Beeinflussung dieser Reaktionen. Ein zweites Themengebiet sind die primären Energie- und Elektrontransferprozesse bei der Photosynthese von Purpurbakterien. Die zentralen Charakteristika dieser ultraschnellen photochemischen Reaktionen (Dynamik und Ausbeute) wurden mittels transienter Absorptionsspektroskopie in Abhängigkeit von äußeren Parametern, wie Temperatur, Anregungswellenlänge und Lösungsmittel, studiert. Hierzu wurde die bestehende Messanlage für Mehrimpulsexperimente ausgebaut und deren Impulserzeugung durch nicht-kollineare optisch parametrische Prozesse auf den nahinfraroten Spektralbereich erweitert. Die Ringschlussreaktion von Indolylfulgiden ist eine ultraschnelle, thermisch nicht aktivierte Reaktion auf der Zeitskala von etwa 0,3 ps. Ihre Quanteneffizienz liegt je nach Substitution der Probe und verwendetem Lösungsmittel zwischen 9 und 20%. Hingegen ist die Ringöffnungsreaktion thermisch aktiviert und verläuft über Barrieren auf der Potentialfläche des ersten elektronisch angeregten Zustands. Ihre Reaktionszeit ist abhängig von Substitution und Lösungsmittel und nimmt Werte zwischen 3 und 16 ps an; die Effizienz liegt im Bereich zwischen 0,1 und 15%. Durch Zuführen von thermischer Energie läuft die Ringöffnungsreaktion schneller und effizienter ab. Ihre Quantenausbeute erhöht sich ebenfalls, wenn man optische Überschussenergie durch Belichtung in die kurzwellige Flanke der Absorptionsbande zur Verfügung stellt. Eine weitere Möglichkeit, die Ringöffnungsquanteneffizienz von Indolylfulgiden zu steigern, ist die Anregung aktiver Schwingungsmoden. Für ein neu synthetisiertes Indolylfulgid mit zwei Cyclopropylringen konnte in einem neuartigen Mehrimpulsexperiment die Ringöffnungsquanteneffizienz nach einer vorgeschalteten Ringschlussreaktion um einen Faktor 3,4 auf annähernd 40% gesteigert werden. Für das Purpurbakterium Rhodobacter sphaeroides wurden verschiedene Energietransferprozesse betrachtet. Zunächst wurde der B800-B850-Transfer in den Antennenkomplexen LH2 mittels einer Mutantenstudie untersucht. Hierzu wurde die Aminosäuresequenz im Bereich der B850-Bindungstasche geändert, was eine verringerte thermische Stabilität und eine Verschiebung der Absorptionsspektren bewirkt. Letztere ist mit einer Verlängerung der Transferzeit von 0,7 ps (Wildtyp) auf bis zu 1,0 ps korreliert. Der Wildtyp – optimiert im Laufe der Evolution – besitzt unter allen untersuchten Proben die beste Funktionalität. In einem weiteren Experiment wurde die ultraschnelle Elektrontransferkaskade im Reaktionszentrum studiert. Über den Zerfall der stimulierten Emission des Special Pair P kann der erste Schritt zum Bakteriochlorophyll BA auf etwa 3,3 ps bestimmt werden. Durch die Beobachtung des transienten Zustands P+BA- wurde die Transferzeit des zweiten Schritts zum Bakteriophäophytin HA zu 0,9 ps ermittelt. An Rhodospirillum rubrum konnte schließlich gezeigt werden, dass die Anregung zwischen Reaktionszentrum und LH1-Antennen sehr schnell äquilibriert und der Elektrontransfer im Reaktionszentrum effektiv mit einer Zeitkonstante von etwa 40 ps abläuft.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
The seasonal culturability (February, April, August) of bacterial cells from a microbial community of an alpine calcareous soil was assessed employing the MicroDrop technique using different laboratory media with humic acid analogs (HA), a mixture of polymers (POL), artificial root exudates (RO), nutrient broth, or soil extract as carbon and energy sources. Thereby, the summer August sample showed the highest culturability value in media supplemented with soil extract (13.5%). Since only 81 wells of a total number of 1008 individual growth tests were overgrown with the February soil sample, the cultivation success was the lowest for the winter environment (0.16%). The major aim of the present study, however, was to assess the cultivation success for cells even exposed to extreme environmental conditions by using defined media. Therefore, subsequent analysis focused on the cultures obtained from the February sample and in media supplemented with RO. It was shown that the monomeric organic carbon of RO proved to be superior to POL and HA for the optimization of the cultivation success (i.e., 71 of the total number of 81 cultures). The quantitative PCR approach confirmed the high coverage of the present analysis since the target groups (Firmicutes, Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Acidobacteria) constituted 73.6% of all eubacteria in the sample whereas the major part was composed of Alphaproteobacteria (49.2%) and Acidobacteria (20.1%). A total of 251 bacteria were analyzed representing 53 distinct phylotypes of which 73% are previously unknown. The majority of the cultured fraction was closely related to the Alphaproteobacteria with the largest number of different phylotypes and the highest evenness value. Although this phylum dominated the cultivated fraction, its cultivation success was hundredfold lower than its abundance in the natural community (0.4% of total cell numbers). Also the Bacteroidetes were most frequently cultured but were dominated by one phylotype (Sphingoterrabacterium pocheensis). The relative culturability of the Bacteroidetes was the highest of all groups and reached 25% of the numbers detected by real-time PCR. The lowest culturability was assessed for the Acidobacteria with only one single cultivated phylotype using media with POL supplemented with signal compounds. However, this phylotype represents a novel, previously unknown acidobacterium, strain Jbg-1. The phylum Acidobacteria mostly consists of environmental 16S rRNA gene sequences and so far comprises only the four validly described species Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present thesis two different novel strains of acidobacteria were isolated. Strain Jbg-1 and the second strain Wbg-1, which was recovered from a coculture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7%, strains Jbg-1 and Wbg-1 are only distantly related to the closest described relative, Terriglobus roseus, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA-similarity between strains Jbg-1 and Wbg-1 of 11.5-13.6% and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. with strain Jbg-1T (= ATCC BAA-1329T = DSM 18101T) as the type strain, and E. aggregans sp. nov. with strain Wbg-1T (= ATCC BAA-1497T = DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions. It was shown that strain Jbg-1 was also well adapted to long-term survival and to higher carbon concentrations after subcultivation. Unexpectedly, a high percentage of interspecific interaction was obtained for the cultivation approach of the February alpine soil (75% cocultures), which represented the major reason for the low cultivation success. Only 16 out of 71 cultures with RO consisted of single cultivated strains. Due to the frequent occurrence of different bacteria in the same cultures, the actual cultivation success was 4.9 fold higher than the value calculated from the abundance of positive cultures. For subsequent analysis, the effect of different treatments during the cultivation approach on the number and composition of bacteria cultured was investigated. In order to differentiate between free-living and attached cells, bacteria were detached from soil particles and used to set up parallel incubations. The detachment from soil particles prior to inoculation had no effect on the total cultivation success and on co-cultivation. Furthermore, signal compounds (cyclic AMP and N-butyryl homoserine lactone), however, increased the cultivation success and co-culturability. Addition of signal compounds yielded different types of activated bacteria and enhanced the total number of phylotypes per co-culture towards 4, 5, 6, and 7 different bacteria. The major part of the single cultivated strains represented a single phylotype, which was related to Sphingoterrabacterium pocheensis. In contrast, most co-cultures contained members of the Alpha- and Betaproteobacteria whereas relatives of Phyllobacterium brassicacearum, Rhodospirillum rubrum, Inqulinus ginsengisoli, Delftia tsuruhatensis, and Rhodocyclus tenuis were the most abundant ones. In conclusion, it is supposed that cell-to-cell interaction routinely occurs between different species of microorganisms, although the way, how these aerobic microorganisms beneficially interact remained to be shown. The elucidation of such interactions seems to be the most successful approach to enhance the culturability of interesting soil bacteria to promote their growth in pure or defined co-cultures.
An electron spin echo modulation frequency analysis of P+-860 is performed in 14N chromatophores of Rhodospirillum rubrum, and in 15N substituted reaction centers from Rhodopseudomonas sphaeroides 2.4.1. For the 14N material two sets of nuclear quadrupole parameters are obtained. From the frequencies found for 15N reaction centers the perpendicular, parallel and isotropic hyperfine previous termcouplingnext term constants of three of the four bacteriochlorophyll nitrogen nuclei are inferred.
The light-induced cation radical of the primary electron donor, Ps70, in photosynthetic reaction centers from Rhodospirillum rubrum G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration. Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for PsO with C2 symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.