Campen, Hanna (2016) The Physiology of Hydrogen Metabolism in Cyanobacteria under Various Environmental Conditions. (Master thesis), Christian-Albrechts-Universität zu Kiel, Kiel, Germany, 85 pp.

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Abstract

This work aimed to investigate the metabolic role of the bidirectional NiFe-hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 considering potential physiological interactions and regulations. The first part of this study focused on the transcriptional regulation of the hoxH gene, encoding for the large subunit of the hydrogenase, and revealed a significant upregulation in cells grown on nitrate-depleted and dissolved organic nitrogen supplemented medium compared to those grown under nitrate enriched conditions. This strongly suggests the hydrogenase to be of high metabolic importance in habitats where nitrate can become limited. Along with results from additional studies, this shows nitrate reduction to be a dominant competitive electron sink of the hydrogenase besides the Calvin-Benson cycle and oxygen. It substantially and further strengthens that senescent phytoplankton blooms, presumably characterized by nitrate depletion and temporary mixotrophic conditions, may represent situations where holding a hydrogenase provides selective advantage. Furthermore, photohydrogen production of the study organism under increasing light intensities was investigated. This was realized by exposing cultures of Synechocystis wild type to five different light intensities (100, 500, 800, 1 OOO and 1400 μmol m-2 s-1) and simultaneously reporting the photohydrogen concentrations produced. Surprisingly, no significant correlation between photo-induced H2 production and the mentioned light intensities was observed. As conditions during dark-light transitions when the bidirectional hydrogenase is active are assumed to be anoxic, major photoprotection mechanisms e.g. the flavoproteins Flvl/Flv3 cannot accept excess electrons since they rely on oxygen as the final acceptor. Therefore, the Flv2-Flv4 dimer proposed to intercept electrons already at the level of PS II is conceivable as a candidate to prevent all electrons from reaching PS I and thereby the hydrogenase. Thus, under light intensities of and above 500 - 800 μmol m-2 s-1, the photosystems probably were already saturated so that a potential correlation between photohydrogen production and light intensities remained masked. Light saturation in the Synechocystis sp. PCC 6803 under the culture conditions in this lab actually seem to set in around 500 - 800 μmol m-2 s-1, as shown by measurements following this work. Potential natural occurrences of the cyanobacterial bidirectional NiFe-hydrogenase were investigated. Therefore, DNA samples were taken at 14 and 6 stations, during two research cruises in the Eastern Tropical Northern Atlantic (ETNA) and the Peruvian upwelling region in the Pacific, respectively. Performing a PCR, the DNA was amplified with l 6S, hypD and hoxH primers to check for the presence of general bacterial DNA, NiFe-hydrogenases of various taxa and of the specific cyanobacterial NiFe-hydrogenase. Since those PCR products could not be sequenced within the time frame of this work, conclusions should be understood as indications only. However, the results generally indicate a rather wide distribution of hydrogenases even in the open Atlantic Ocean (> 50% potentially hoxH positive), considered being extensively nutrient-depleted and therefore characterized by rather low primary production. Possibly, nutrients transported offshore by so-called eddies partially allow phytoplankton blooms and may provide an explanation for indicated potential hoxH occurrences. In the Peruvian upwelling region, stratification might have been the critical factor for phytoplankton bloom establishment correlating with indicated potential hoxH presence at three stations, and anti-correlating with recent upwelling events at two stations where hoxH was potentially absent. Stratification and continuous production presumably led to nitrate depletion as well as mixotrophic conditions at potentially hoxH positive stations, pronouncing the idea, that hydrogenases could be selectively advantageous here.

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