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Müller, Johannes (2012): Interspecies interaction and diversity of green sulfur bacteria. Dissertation, LMU München: Faculty of Biology



The following work is shedding light on the phylogenetic classification on the family of the Chlorobiacea, the members of which are showing signs of preadaptation to symbiosis. Symbioses consisting of purely prokaryotic associations between phylogenetically distinct bacterial species have been widely documented. Only few are available as a laboratory culture to elucidate the molecular basis of their interaction. One of these few model organisms is the phototrophic consortium “Chlorochromatium aggregatum”. It consists of 12-20 green sulfur bacteria epibionts surrounding a central, Betaproteobacterium in a highly ordered fashion. The phototrophic partner bacterium, belonging to the green sulfur bacteria, is available in pure culture and its physiology has been studied in detail. In this work, novel insights into the physiology of the central bacterium that was previously uncharacterized are provided. The family of the Chlorobiaceae represents a phylogenetically coherent and isolated group within the domain Bacteria. Green sulfur bacteria are obligate photolithoautotrophs that require highly reducing conditions for growth and can utilize only a very limited number of carbon substrates. These bacteria thus inhabit a very narrow ecologic niche. For the phylogenetic studies on green sulfur bacteria, 323 16S rRNA gene sequences, including cultured species as well as environmental sequences were analysed. By rarefaction analysis and statistical projection, it was shown that the data represent nearly the whole spectrum of green sulfur bacterial species that can be found in the sampled habitats. Sequences of cultured species, however, did not even cover half of the biodiversity. In the 16S rDNA gene tree, different clusters were found that in most cases correlated with physiological adaptations of the included species. By combining all sampling sites of green sulfur bacteria in a world map, large, unsampled areas were revealed and it could be shown that in some regions, a non-random distribution of GSB occurred. The wide dispersal of green sulfur bacterial species can be seen in sequences that were found ubiquitously all over the world. To imitate the phylogenetical relationships of whole genome analyses, a concatenated tree was constructed including 32 species and 3 different genetic regions, the bchG gene, the sigA gene and the fmoA gene. Comparison with the 16S rRNA gene tree showed more genetic differences between species, and led to a higher resolution and a more dependable phylogeny. A distance matrix comparison showed that the fmoA gene sequence has the highest correlation to the 16S rDNA of the sequences investigated. Additionally, a dissimilarity matrix revealed that the fmoA gene sequence provides the highest phylogenetic resolution among the sequences investigated. Therefore, we showed that the fmoA gene sequence is the most suitable among the sequences investigated to support the 16S rDNA phylogeny of green sulfur bacteria. To overcome the limitation of immobility, some green sulfur bacteria have entered into a symbiosis with motile Betaproteobacteria in a type of multicelllular association termed phototrophic consortia. Recent genomic, transcriptomic, and proteomic studies of "C. aggregatum" and its epibiont provided insights into the molecular basis and the origin of the stable association between the two very distantly related bacteria. However, to date the possibility of a metabolic coupling between the bacterial partners has not been investigated. The symbiotic exchange of metabolites between the two species was therefore investigated by tracking the flux of isotope-labeled CO2 through the two partner organisms using NanoSIMS analysis and magnetic capture, revealing a fast and simultaneous incorporation of labeled carbon into both organisms. The transferred metabolites were identified by isotopologue profiling for which the partner cells were separated by cesium chloride density gradient centrifugation, a method which identified amino acids as one group of substrates to be transferred between the two partners. The addition of external carbon substrates inhibited the transfer between the two partners, suggesting that transporters are the means by which substrates are exchanged. Genome sequencing revealed the central bacterium to be an aerobic or microaerophilic chemoheterotrophic bacterium. The existence of 32 PAS domains which are responsible for sensing various signals indicate that the central bacterium is responsible for the chemo- and phototactic responses of the consortium. The central bacterium possesses all traits of an autonomous organism. However, transcriptome analysis revealed the central bacterium to be inactive in the dark although external carbon sources were present. Thereby, a yet unexplained dependence on the epibiont is revealed which indicates a complex metabolic coupling between the two symbiotic partner organisms.