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Glaeser, Jens (2004): In situ Metabolism and Biogeography of Phototrophic Consortia. Dissertation, LMU München: Faculty of Biology
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Abstract

>>Novel bacteriochlorophyll e structures and species-specific variability of pigment composition in green sulfur bacteria<< The relative composition of bacteriochlorophyll (BChl) homologs in five different strains of brown-colored green sulfur bacteria was investigated by HPLC-MS/MS and NMR analyses. In addition, the effect of incubation light intensities on homolog distribution was studied in one of the strains (strain Dagow III). A total of 23 different BChl e structures were detected and comprise four homologous porphyrin ring systems and eight different esterifying alcohols. Several BChl e structures are novel. These include a C-8 ethyl, C-12 methyl [E,M] BChl eF homolog which was identified by 1H-NMR analyses of the isolated, main farnesyl homologs (BChl eF). In addition, five previously unknown homolog series with dodecanol, pentadecenol, tetradecanol, hexadecenol and phytol as the esterifying alcohols were detected. The composition of BChl e homologs from the five strains of green sulfur bacteria differed with respect to the relative abundance of the homologs (BChl eF: 25.6 to 67.0% of total BChl e content in stationary cultures). In strain Dagow III, the abundance of BChl eF homologs decreased upon entry into the stationary phase. In all free-living strains, the abundance of BChl eF was increased when the relative carotenoid content was low. The present results provide a more detailed picture of pigment composition in chlorosomes and thus will help to elucidate their structure and function. Furthermore, the newly discovered BChl e molecules are valuable biomarkers for the study of the occurrence and the metabolism of green sulfur bacteria in past and present ecosystems. >>Characterization and in situ carbon metabolism of phototrophic consortia<< A dense population of the phototrophic consortium "P. roseum" was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescent in situ hybridization revealed that the brown-colored epibionts of "P. roseum" constituted up to 37 % of total bacterial cell numbers and up to 88 % of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and DGGE fingerprinting yielded a maximum of 4 different DNA bands depending on the year of study, indicating a low diversity of green sulfur bacteria. The 465 bp-long 16S rRNA gene sequence of the epibiont of "P. roseum" was obtained after sorting individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. A maximum of light-dependent H14CO3- fixation in the chemocline in the presence of DCMU suggested anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determination of stable isotope ratios (d13C) of their specific biomarkers. Analysis of photosynthetic pigments by HPLC revealed the presence of high concentrations of BChl e and smaller amounts of BChl a, d and Chl a in the chemocline. Unexpectedly, isorenieratene and b-isorenieratene, carotenoids typical for other brown-colored members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their d13C measured. Farnesol, tetradecanol, hexadecanol and hexadecenol all were significantly enriched in 13C compared to bulk dissolved and particulate organic carbon, and compared to the biomarkers of purple sulfur bacteria. The difference (Dd13C) between d13C values of farnesol, the major esterifying alcohol of BChl e, and CO2 was -7.1‰, which provides clear evidence for a photoautotrophic mode of growth of the green sulfur bacterial epibionts of "P. roseum" in situ. >>The significance of organic carbon compounds for in situ metabolism and chemotaxis of phototrophic consortia<< The significance of organic carbon substrates for the chemotaxis and physiology of phototrophic consortia was investigated in a dense chemocline community of "Pelochromatium roseum". For the first time, the monopolar monotrichous flagellation of the central bacterium could be visualized. In situ, intact motile "P. roseum" consortia were strongly attracted by sulfide and 2-oxoglutarate, which indicated a potential role of these compounds in the metabolism of "P. roseum". In chemocline water samples, 2-[14C(U)]-oxoglutarate was utilized at nanomolar concentrations (half saturation constant of uptake Kt ≤ 10 - 40 nM), and at a maximum uptake rate of Vmax ≈ 6 nM·h-1. The calculated turnover of 2-oxoglutarate at in situ concentrations was ~6 hours. Microautoradiography of chemocline water samples revealed that 87.5 % of the "P. roseum" consortia incorporated 2-oxoglutarate when both light and sulfide were present, whereas uptake was detected in less than 1.4% of the consortia if either light or sulfide were absent. Since the green sulfur bacterial epibionts in "P. roseum" have been shown to grow autotrophically, 2-oxoglutarate most likely is taken up and utilized by the central bacterium. Thus, our results indicate that incorporation of 2-oxoglutarate by the central bacterium is regulated by the metabolic state of the green sulfur bacterial epibionts. Diversity and biogeography of phototrophic consortia Motile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bacteria surround a flagellated colorless b-proteobacterium in the center. To date, seven different morphological types have been described. In addition, two immotile associations involving green sulfur bacteria are known. Employing a culture-independent approach, different types of phototrophic consortia were mechanically isolated by micromanipulation from 14 freshwater lakes and ponds and partial 16S rRNA gene sequences of the green sulfur bacterial epibionts were determined. In all cases, phototrophic consortia with the same morphology and from the same habitat contained only one single epibiont phylotype. None of the epibiont 16S rRNA gene sequences have so far been detected in free-living green sulfur bacteria, indicating that the interaction between epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate one. Morphologically indistinguishable phototrophic consortia collected from different lakes contained different epibionts. Overall, 20 different types of epibionts were detected in the present study. Based on our phylogenetic analysis, the epibiont sequences are not monophyletic and arose from different ancestors which independently developed the ability to form symbiotic associations. In the majority of lakes investigated, different types of phototrophic consortia were found to co-occur. Based on this finding, morphologically distinct types of consortia occupy different ecological niches. This is supported by the results of chemotaxis assays which revealed that different populations of the consortia "Chlorochromatium aggregatum" and "Pelochromatium roseum" were attracted by different organic carbon substrates and different reduced sulfur compounds. The present study thus demonstrates a high diversity, the presence of multiple niches, and a nonstatistical biogeographical distribution of phototrophic consortia in the natural environment.