Copper is a trace element utilized by organisms as a cofactor involved in redox chemistry, electron transport, photosynthesis, and oxidation reactions. In excess, copper is toxic; it can generate reactive oxygen species causing cellular damage, or poison other metalloproteins by replacing native metal cofactors. Gram-negative bacteria have developed homeostatic mechanisms to maintain the intracellular copper concentration in the face of changing environmental conditions. For Gram-negative enteric bacteria, like Esherichiacoli and Salmonella enterica serovar typhimurium, copper is encountered in industrial and institutional settings, where the metal is used as a broad-spectrum biocide. For environmental bacteria, such as the marine cyanobacterium Synechococcus sp. WH8102, copper stress occurs because human activity changes the concentration of copper in the ocean. This dissertation contains six chapters, relating four stories of our investigations into the molecular mechanisms of copper homeostasis in Gram-negative bacteria. Chapter I contains literature review and background on the implications of bacterial copper homeostasis. Chapter II reports our work investigating the expression of two E. coli proteins, CusF and CusB, upon copper stress; we show that CusF expresses at a ~10-fold molar excess over CusB. Chapter III describes a collaboration between our lab and Jose Argüello's lab at Worcester Polytechnic Institute, and we show that CusF can acquire Cu(I) from CopA. Our results from Chapters II and III show that CusF functions as a major copper chaperone in the periplasm of E. coli. Chapter IV details our work characterizing a novel protein from marine cyanobacteria, Synw_0921. Although Synw_0921 is believed to be involved in copper homeostasis, we show that it is an iron-sulfur cluster protein. Bioinformatic analysis suggests that Synw_0921 represents a new family of proteins that help marine cyanobacteria adapt to copper changes in their unique environment. Chapter V relates our work on CueR and GolS, two homologous sensor proteins with distinct metal-dependent transcriptional activation; we find that the activity cannot be explained by binding affinity differences. Chapter VI concludes with final thoughts on the intersection of biochemistry and molecular biology in the important process of understanding copper homeostasis.

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In a climate change perspective, increased air temperatures are already a reality and are expected to increase even more in the future, especially in areas at high latitudes. The present thesis therefore addresses the influence of climate change on the physical properties and the phytoplankton communities of typical small and oligotrophic lakes in northern Sweden (62-64˚N). In the first part of the study, we found a significant trend (10 lakes from 1916 to 2010) of ice break-ups occurring increasingly earlier. The timing of ice break-up was strongly influenced by the April air temperature indicating that expected increases in air temperature in the future will also result in an earlier ice break-up. We also used concentrations of chlorophyll a (chl a) as estimations of phytoplankton biomass and discovered a positive relationship between surface water temperature and concentrations of chl a in Lake Remmaren (from 1991 to 2008). The second part of the thesis focuses on climatic conditions and cyanobacteria abundance in three small, oligotrophic lakes in northern Sweden; Lake Remmaren, Lake S. Bergsjön and Lake Gransjön. The concentration and relative abundance of cyanobacteria differ between 2011 and 2012, with different climatic conditions. The "warm" year of 2011 had higher concentrations and relative abundance of cyanobacteria than the "cold" year of 2012. Trends in increasing surface water temperatures as well as increasing abundance of cyanobacteria in August were found in Lake Remmaren (from 1988 to 2011). The direct or indirect effects of warming had a positive effect on the cyanobacteria abundance, since nutrients (Tot N and Tot P) did not display an increasing trend in Lake Remmaren. An analysis on the composition of phytoplankton species in Lake Remmaren, Lake S. Bergsjön and Lake Gransjön revealed that the cyanobacteria Merismopedia sp. was more common in 2011 than 2012. If different cyanobacteria become more common in oligotrophic lakes in the future, the functioning of lake ecosystems may be impacted. Small zooplankton eats small phytoplankton and if smaller phytoplankton species, e.g. cyanobacteria, increase at the expense of other phytoplankton groups, an extra step in the food chain might be added. Less energy might be transferred to the upper levels because many cyanobacteria contain toxic compounds and are less edible than other phytoplankton groups. An increase of toxic containing cyanobacteria in lakes can also make lakes less attractive for recreational purposes in the future.

A hybrid vector, pBVl, has been constructed which is capable of regulating expression of cloned genes in both Escherichia coli and in cyanobacterium Synechocystis 6803. Vector pBVl contains the powerful rightward promoter of bacteriophage lambda to ensure that cloned genes are transcibed at a high level. In addition, pBV] also contains a gene, cI857, encoding the lambda temperature sensitive repressor protein.The CAT gene coding for chloramphenicol aceyltransferase (CmActase) was cloned into pBV1 downstream of the lambda regulatory features to make plasmid pTC1. The expression of the CAT gene was quantified spectrophotometrically following addition of chloramphenicol and a shift in the growth temperature of the cell culture. The specific activity of CmActase increased from 540 to 5400 units within 30 minutes. Thus, using the newly constructed hybrid vector, pBVl, temperature regulation of gene expression in E. coli was observed. / Department of Biology

A hybrid vector, pANIC1, was to be constructed which was capable of regulating expression of cloned genes in both Escherichia coli and Anacystis nidulans R2 by controlling the iron content of the growth medium. Plasmid pANIC1 would have origins of replication for E. coli and A. nidulans R2, and a marker gene conferring ampicillin resistance. It would also contain the promoter for the irpA gene which is active only in low iron growth conditions.The first two stages of the construction were successfully completed, but unfortunately the final construction proved to be unstable. Recent information has shown that operator sequences upstream from the irpA gene's promoter result in an unstable message. This may be interfering with the normal functioning of the host cell, resulting in an unstable construction. In future experiments it may be neccessary to alter the growth conditions or remove the upstream sequences in order to stablize the construction. / Department of Biology

Photosynthetic membranes were prepared from the cyanobacterium Synechococcus PCC 7942 with oxygen evolving specific activity of 250-300 µmoles 02/ mg chl/hr. The membranes retained activity with a half-life of 4-5 days when stored at 0°C, or when quickly frozen in liquid nitrogen, greater than 95% of the activity remained after 2 months. Attempts to purify homogeneous preparations of photosystem II complexes from these membranes by detergent extraction were unsuccessful as indicated by a lack of a significant increase in oxygen evolution specific activity of the detergent extracts. Photosynthetic membrane detergent extracts usually maintained the same oxygen evolution specific activity as the orginal membranes, and a considerable amount of Photosystem I activity (75 µmoles 02 consumed /mg chl/hr in the Mehler reaction) was still present. The donor side of the photosystem II particles in the detergent extract was intact since the artificial electron acceptor, 2,6-dichiorophenolindophenol (DCPIP), was reduced at a rate comparable to the oxygen evolving activity. All oxygen evolving activity of the detergent extracts was lost when ion-exchange chromatography was used to resolve the co-extracted photosystem II and photosystem I complexes. / Department of Biology

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites.

Bibliography: leaves 252-267. / xvi, 267 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / These cyanobacteria are two freshwater phytoplankton which produce toxins, taint the taste and odour of potable water and form surface blooms. A technique was developed to assess cell metabolic activity and to determine the influence of light and nutrients on gas reside synthesis and bouyancy regulation in order to understand factors which favour their dominance, and thus how to control them. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1998?

Bibliography: leaves 235-265. / xvi, 265 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the tumour promoting effects of the microcystins through a long-term study in which cyanobacterial extract containing a range of microcystins was given in drinking water to mice previously treated with the tumour initiator N-nitroso-N-methyluren by gavage ; and through examining the effects of pure microcystin-LR in cultured primary hepatocytes from immature mice. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1998?